U.S. patent number 7,241,879 [Application Number 10/899,715] was granted by the patent office on 2007-07-10 for immunoassays for anti-hcv antibodies.
This patent grant is currently assigned to Novartis Vaccines and Diagnostics, Inc.. Invention is credited to Phillip Arcangel, David Y. Chien, Doris Coit, Carlos George-Nascimento, Angelica Medina-Selby, Laura Tandeske.
United States Patent |
7,241,879 |
Chien , et al. |
July 10, 2007 |
Immunoassays for anti-HCV antibodies
Abstract
HCV immunoassays comprising an NS3/4a conformational epitope and
a multiple epitope fusion antigen are provided, as well as
immunoassay solid supports for use with the immunoassays.
Inventors: |
Chien; David Y. (Alamo, CA),
Arcangel; Phillip (Oakland, CA), Tandeske; Laura (San
Leandro, CA), George-Nascimento; Carlos (Walnut Creek,
CA), Coit; Doris (Petaluma, CA), Medina-Selby;
Angelica (San Francisco, CA) |
Assignee: |
Novartis Vaccines and Diagnostics,
Inc. (Emeryville, CA)
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Family
ID: |
27395684 |
Appl.
No.: |
10/899,715 |
Filed: |
July 26, 2004 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20040265801 A1 |
Dec 30, 2004 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10637323 |
Aug 8, 2003 |
6797809 |
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09881654 |
Jun 14, 2001 |
6632601 |
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60280867 |
Apr 2, 2001 |
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60280811 |
Apr 2, 2001 |
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60212082 |
Jun 15, 2000 |
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Current U.S.
Class: |
536/23.4;
435/69.7; 530/350; 435/328 |
Current CPC
Class: |
G01N
33/5767 (20130101); C07K 14/005 (20130101); G01N
2469/10 (20130101); C07K 2319/00 (20130101); G01N
2469/20 (20130101); G01N 2333/18 (20130101); C12N
2770/24222 (20130101) |
Current International
Class: |
C07H
21/04 (20060101) |
Field of
Search: |
;530/350
;435/41,69.7,328 ;536/23.4 |
References Cited
[Referenced By]
U.S. Patent Documents
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5350671 |
September 1994 |
Houghton et al. |
5683864 |
November 1997 |
Houghton et al. |
5712087 |
January 1998 |
Houghton et al. |
5843752 |
December 1998 |
Dasmahapatra et al. |
5871904 |
February 1999 |
Kashiwakuma et al. |
5990276 |
November 1999 |
Zhang et al. |
6171782 |
January 2001 |
Houghton et al. |
6428792 |
August 2002 |
Valenzuela et al. |
6514731 |
February 2003 |
Valenzuela et al. |
6630298 |
October 2003 |
Chien et al. |
2003/0044774 |
March 2003 |
Valenzuela et al. |
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318216 |
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388232 |
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EP |
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0450931 |
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EP |
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0472207 |
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EP |
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0 870 830 |
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Oct 1998 |
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EP |
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2 212 511 |
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Jul 1989 |
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GB |
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2 257 784 |
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Jan 1993 |
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GB |
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97 247 |
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Jul 1997 |
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PT |
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WO 93/00365 |
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Jan 1993 |
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WO |
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WO 94/01778 |
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Jan 1994 |
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WO |
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WO 94 25601 |
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Nov 1994 |
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WO |
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WO 97 12043 |
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Apr 1997 |
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WO |
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WO 97/44469 |
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Nov 1997 |
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WO |
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Other References
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pool-screening systems," Transfusion 40:575-579, 2000. cited by
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the ATPase/helicase domain, but not the protease domain of the
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Gastroenterology Hepatology 8:S33-39 (1993). cited by other .
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antibodies directed to the putative core, NS4, and NS5 region
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Implications for Diagnos, Development and Control of Viral
Disease," Hepatology 14:381-388 (1991). cited by other .
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ELISA using a synthetic peptide comprising a structural epitope,"
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Etiologic Virus of Human Non-A, Non-B Hepatitis," Science
244:362-364 (1989). cited by other .
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Libraries and Improved Diagnosis with a Chimeric Antigen," J.
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cited by other.
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Primary Examiner: Foley; Shanon
Assistant Examiner: Le; Emily M.
Attorney, Agent or Firm: Lillis; Marcella Robins; Roberta L.
Harbin; Alisa A.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation application of U.S. patent
application Ser. No. 10/637,323, filed Aug. 8, 2003, now U.S. Pat.
No. 6,797,809, which is a divisional of U.S. patent application
Ser. No. 09/881,654, filed Jun. 14, 2001, now U.S. Pat. No.
6,632,601, from which applications priority is claimed pursuant to
35 USC .sctn.120 and which applications are related to provisional
patent application Ser. Nos. 60/212,082, filed Jun. 15, 2000;
60/280,811, filed Apr. 2, 2001; and 60/280,867, filed Apr. 2, 2001,
from which applications priority is claimed under 35 USC
.sctn.119(e)(1) and which applications are incorporated herein by
reference in their entireties.
Claims
The invention claimed is:
1. A polynucleotide comprising a coding sequence for a multiple
epitope fusion antigen, wherein said multiple epitope fusion
antigen the amino acid sequence of SEQ ID NO: 4, or an amino acid
sequence with at least 80% sequence identity thereto which binds
specifically with anti-HCV antibodies present in a biological
sample from an HCV-infected individual, wherein said antibodies
specifically bind with SEQ ID NO: 4.
2. The polynucleotide of claim 1, wherein said polynucleotide
comprises a coding sequence for a multiple epitope fusion antigen
that comprises an amino acid sequence with at least 90% sequence
identity to the amino acid sequence of SEQ ID NO: 4 which binds
specifically with anti-HCV antibodies present in a biological
sample from an HCV-infected individual, wherein said antibodies
specifically bind with SEQ ID NO: 4.
3. The polynucleotide of claim 1, wherein said polynucleotide
comprises a coding sequence for a multiple epitope fusion antigen
that comprises an amino acid sequence with at least 98% sequence
identity to the amino acid sequence of SEQ ID NO: 4, which binds
specifically with anti-HCV antibodies present in a biological
sample from an HCV-infected individual, wherein said antibodies
specifically bind with SEQ ID NO: 4.
4. The polynucleotide of claim 1, wherein said polynucleotide
comprises a coding sequence for a multiple epitope fusion antigen
that consists of the amino acid sequence of SEQ ID NO: 4.
5. The polynucleotide of claim 1, wherein said polynucleotide
comprises the nucleic acid sequence of SEQ ID NO: 3, or a nucleic
acid sequence with at least 80% sequence identity thereto which
encodes a polypeptide that binds specifically with anti-HCV
antibodies present in a biological sample from an HCV-infected
individual.
6. The polynucleotide of claim 1, wherein said polynucleotide
comprises the nucleic acid sequence of SEQ ID NO: 3, or a nucleic
acid sequence with at least 90% sequence identity thereto which
encodes a polypeptide that binds specifically with anti-HCV
antibodies present in a biological sample from an HCV-infected
individual.
7. The polynucleotide of claim 1, wherein said polynucleotide
comprises the nucleic acid sequence of SEQ ID NO: 3, or a nucleic
acid sequence with at least 98% sequence identity thereto which
encodes a polypeptide that binds specifically with anti-HCV
antibodies present in a biological sample from an HCV-infected
individual.
8. A recombinant vector comprising: (a) a polynucleotide according
to claim 1; (b) and control elements operably linked to said
polynucleotide whereby the coding sequence can be transcribed and
translated in a host cell.
9. A recombinant vector comprising: (a) a polynucleotide according
to claim 4; (b) and control elements operably linked to said
polynucleotide whereby the coding sequence can be transcribed and
translated in a host cell.
10. A recombinant vector comprising: (a) a polynucleotide according
to claim 5; (b) and control elements operably linked to said
polynucleotide whereby the coding sequence can be transcribed and
translated in a host cell.
11. A recombinant vector comprising: (a) a polynucleotide according
to claim 7; (b) and control elements operably linked to said
polynucleotide whereby the coding sequence can be transcribed and
translated in a host cell.
12. A host cell transformed with the recombinant vector of claim
8.
13. A host cell transformed with the recombinant vector of claim
9.
14. A host cell transformed with the recombinant vector of claim
10.
15. A host cell transformed with the recombinant vector of claim
11.
16. A method of producing a recombinant multiple epitope fusion
antigen comprising: (a) providing a population of host cells
according to claim 12; and (b) culturing said population of cells
under conditions whereby the multiple epitope fusion antigen
encoded by the coding sequence present in said recombinant vector
is expressed.
17. A method of producing a recombinant multiple epitope fusion
antigen comprising: (a) providing a population of host cells
according to claim 13; and (b) culturing said population of cells
under conditions whereby the multiple epitope fusion antigen
encoded by the coding sequence present in said recombinant vector
is expressed.
18. A method of producing a recombinant multiple epitope fusion
antigen comprising: (a) providing a population of host cells
according to claim 14; and (b) culturing said population of cells
under conditions whereby the multiple epitope fusion antigen
encoded by the coding sequence present in said recombinant vector
is expressed.
19. A method of producing a recombinant multiple epitope fusion
antigen comprising: (a) providing a population of host cells
according to claim 15; and (b) culturing said population of cells
under conditions whereby the multiple epitope fusion antigen
encoded by the coding sequence present in said recombinant vector
is expressed.
Description
TECHNICAL FIELD
The present invention pertains generally to viral diagnostics. In
particular, the invention relates to immunoassays using multiple
HCV antigens, for accurately diagnosing hepatitis C virus
infection.
BACKGROUND OF THE INVENTION
Hepatitis C Virus (HCV) is the principal cause of parenteral non-A,
non-B hepatitis (NANBH) which is transmitted largely through body
blood transfusion and body fluid exchange. The virus is present in
0.4 to 2.0% of the general population in the United States. Chronic
hepatitis develops in about 50% of infections and of these,
approximately 20% of infected individuals develop liver cirrhosis
which sometimes leads to hepatocellular carcinoma. Accordingly, the
study and control of the disease is of medical importance.
HCV was first identified and characterized as a cause of NANBH by
Houghten et al. The viral genomic sequence of HCV is known, as are
methods for obtaining the sequence. See, e.g., International
Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. HCV has
a 9.5 kb positive-sense, single-stranded RNA genome and is a member
of the Flaviridae family of viruses. At least six distinct, but
related genotypes of HCV, based on phylogenetic analyses, have been
identified (Simmonds et al., J. Gen. Virol. (1993) 74:2391 2399).
The virus encodes a single polyprotein having more than 3000 amino
acid residues (Choo et al., Science (1989) 244:359 362; Choo et
al., Proc. Natl. Acad. Sci. USA (1991) 88:2451 2455; Han et al.,
Proc. Natl. Acad. Sci. USA (1991) 88:1711 1715). The polyprotein is
processed co- and post-translationally into both structural and
non-structural (NS) proteins.
In particular, as shown in FIG. 1, several proteins are encoded by
the HCV genome. The order and nomenclature of the cleavage products
of the HCV polyprotein is as follows:
NH.sub.2-C-E1-E2-P7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Initial
cleavage of the polyprotein is catalyzed by host proteases which
liberate three structural proteins, the N-terminal nucleocapsid
protein (termed "core") and two envelope glycoproteins, "E1" (also
known as E) and "E2" (also known as E2/NS1), as well as
nonstructural (NS) proteins that contain the viral enzymes. The NS
regions are termed NS2, NS3, NS4, NS4a, NS4b, NS5a and NS5b. NS2 is
an integral membrane protein with proteolytic activity. NS2, either
alone or in combination with NS3, cleaves the NS2-NS3 sissle bond
which in turn generates the NS3 N-terminus and releases a large
polyprotein that includes both serine protease and RNA helicase
activities. The NS3 protease serves to process the remaining
polyprotein. Completion of polyprotein maturation is initiated by
autocatalytic cleavage at the NS3-NS4a junction, catalyzed by the
NS3 serine protease. Subsequent NS3-mediated cleavages of the HCV
polyprotein appear to involve recognition of polyprotein cleavage
junctions by an NS3 molecule of another polypeptide. In these
reactions, NS3 liberates an NS3 cofactor (NS4a), NS4b and NS5a
(NS5A has a phosphorylation function), and an RNA-dependent RNA
polymerase (NS5b).
A number of general and specific polypeptides useful as
immunological and diagnostic reagents for HCV, derived from the HCV
polyprotein, have been described. See, e.g., Houghton et al.,
European Publication Nos. 318,216 and 388,232; Choo et al., Science
(1989) 244:359 362; Kuo et al., Science (1989) 244:362 364;
Houghton et al., Hepatology (1991) 14:381 388; Chien et al., Proc.
Natl. Acad. Sci. USA (1992) 89:10011 10015; Chien et al., J.
Gastroent. Hepatol. (1993) 8:S33 39; Chien et al., International
Publication No. WO 93/00365; Chien, D. Y., International
Publication No. WO 94/01778. These publications provide an
extensive background on HCV generally, as well as on the
manufacture and uses of HCV polypeptide immunological reagents. For
brevity, therefore, the disclosure of these publications is
incorporated herein by reference.
Sensitive, specific methods for screening and identifying carriers
of HCV and HCV-contaminated blood or blood products would provide
an important advance in medicine. Post-transfusion hepatitis (PTH)
occurs in approximately 10% of transfused patients, and HCV has
accounted for up to 90% of these cases. Patient care as well as the
prevention and transmission of HCV by blood and blood products or
by close personal contact require reliable diagnostic and
prognostic tools. Accordingly, several assays have been developed
for the serodiagnosis of HCV infection. See, e.g., Choo et al.,
Science (1989) 244:359 362; Kuo et al., Science (1989) 244:362 364;
Choo et al., Br. Med. Bull. (1990) 46:423 441; Ebeling et al.,
Lancet (1990) 335:982 983; van der Poel et al., Lancet (1990)
335:558 560; van der Poel et al., Lancet (1991) 337:317 319; Chien,
D. Y., International Publication No. WO 94/01778; Valenzuela et
al., International Publication No. WO 97/44469; and Kashiwakuma et
al., U.S. Pat. No. 5,871,904.
A significant problem encountered with some serum-based assays is
that there is a significant gap between infection and detection of
the virus, often exceeding 80 days. This assay gap may create great
risk for blood transfusion recipients. To overcome this problem,
nucleic acid-based tests (NAT) that detect viral RNA directly, and
HCV core antigen tests that assay viral antigen instead of antibody
response, have been developed. See, e.g., Kashiwakuma et al., U.S.
Pat. No. 5,871,904.
However, there remains a need for sensitive, accurate diagnostic
and prognostic tools in order to provide adequate patient care as
well as to prevent transmission of HCV by blood and blood products
or by close personal contact.
SUMMARY OF THE INVENTION
The present invention is based in part, on the finding that the use
of NS3/4a conformational epitopes, in combination with multiple
epitope fusion antigens, provides a sensitive and reliable method
for detecting early HCV seroconversion. The assays described herein
can also detect HCV infection caused by any of the six known
genotypes of HCV. The use of multiple epitope fusion proteins also
has the added advantages of decreasing masking problems, improving
sensitivity in detecting antibodies by allowing a greater number of
epitopes on a unit area of substrate, and improving
selectivity.
Accordingly, in one embodiment, the subject invention is directed
to an immunoassay solid support consisting essentially of at least
one HCV NS3/4a conformational epitope and a multiple epitope fusion
antigen, bound thereto, wherein said NS3/4a epitope and/or said
multiple epitope fusion antigen react specifically with anti-HCV
antibodies present in a biological sample from an HCV-infected
individual.
The NS3/4a epitope may comprise the amino acid sequence depicted in
FIGS. 3A 3D, or an amino acid sequence with at least 80% sequence
identity thereto, or 90% sequence identity thereto, or at least 98%
sequence identity thereto, or any integer in between, so long as
the sequence has protease activity. In certain embodiments, the
NS3/4a conformational epitope consists of the amino acid sequence
depicted in FIGS. 3A 3D.
In additional embodiments, the multiple epitope fusion antigen
comprises the amino acid sequence depicted in FIGS. 5A 5F, or an
amino acid sequence with at least 80% sequence identity thereto, or
90% sequence identity thereto, or at least 98% sequence identity
thereto, or any integer in between, so long as the sequence reacts
specifically with anti-HCV antibodies present in a biological
sample from an HCV-infected individual. In certain embodiments, the
multiple epitope fusion antigen consists of the amino acid sequence
depicted in FIGS. 5A 5F.
In yet another embodiment, the subject invention is directed to an
immunoassay solid support consisting essentially of at least one
HCV NS3/4a conformational epitope and a multiple epitope fusion
antigen, bound thereto, wherein said NS3/4a conformational epitope
comprises the amino acid sequence depicted in FIGS. 3A 3D, or an
amino acid sequence with at least 80% sequence identity thereto
which has protease activity, and said multiple epitope fusion
antigen comprises the amino acid sequence depicted in FIGS. 5A 5F,
or an amino acid sequence with at least 80% sequence identity,
thereto which reacts specifically with anti-HCV antibodies present
in a biological sample from an HCV-infected individual. In certain
embodiments, the NS3/4a conformational epitope and the multiple
epitope fusion antigen have at least 90%, 98% (or any integer
between) sequence identity to the amino acid sequences of FIGS. 3A
3D and FIGS. 5A 5F, respectively, so long as the NS3/4a sequence
has protease activity, and the multiple epitope fusion antigen
reacts specifically with anti-HCV antibodies present in a
biological sample from an HCV-infected individual. In certain
embodiments, the NS3/4a conformational epitope consists of the
amino acid sequence depicted in FIGS. 3A 3D, and the multiple
epitope fusion antigen consists of the amino acid sequence depicted
in FIGS. 5A 5F.
In another embodiment, the invention is directed to an immunoassay
solid support consisting essentially of at least one HCV NS3/4a
conformational epitope and a multiple epitope fusion antigen, bound
thereto, wherein said NS3/4a conformational epitope consists of the
amino acid sequence depicted in FIGS. 3A 3D, and said multiple
epitope fusion antigen consists of the amino acid sequence depicted
in FIGS. 5A 5F.
In still a further embodiment, the invention is directed to a
method of detecting hepatitis C virus (HCV) infection in a
biological sample, said method comprising:
(a) providing an immunoassay solid support as described above;
(b) combining a biological sample with said solid support under
conditions which allow HCV antibodies, when present in the
biological sample, to bind to said NS3/4a epitope and/or said
multiple epitope fusion antigen to form a first immune complex;
(c) adding to the solid support from step (b) under complex forming
conditions a detectably labeled antibody, wherein said labeled
antibody is reactive with said immune complex;
(d) detecting second immune complexes formed between the detectably
labeled antibody and the first immune complex, if any, as an
indication of HCV infection in the biological sample.
In still a further embodiment, the invention is directed to a
method of detecting hepatitis C virus (HCV) infection in a
biological sample, said method comprising:
(a) providing an immunoassay solid support consisting essentially
of at least one HCV NS3/4a conformational epitope and a multiple
epitope fusion antigen, bound thereto, wherein said NS3/4a
conformational epitope consists of the amino acid sequence depicted
in FIGS. 3A 3D, and said multiple epitope fusion antigen consists
of the amino acid sequence depicted in FIGS. 5A 5F;
(b) combining a biological sample with said solid support under
conditions which allow HCV antibodies, when present in the
biological sample, to bind to said NS3/4a epitope and/or said
multiple epitope fusion antigen to form a first immune complex;
(c) adding to the solid support from step (b) under complex forming
conditions a detectably labeled antibody, wherein said labeled
antibody is reactive with said immune complex;
(d) detecting second immune complexes formed between the detectably
labeled antibody and the first immune complex, if any, as an
indication of HCV infection in the biological sample.
In another embodiment, the invention is directed to an
immunodiagnostic test kit comprising an immunoassay solid support
as described above, and instructions for conducting the
immunodiagnostic test.
In another embodiment, the subject invention is directed to a
method of producing an immunoassay solid support, comprising:
(a) providing a solid support; and
(b) binding to the solid support at least one HCV NS3/4a
conformational epitope and a multiple epitope fusion antigen,
wherein said NS3/4a epitope and/or said multiple epitope fusion
antigen react specifically with anti-HCV antibodies present in a
biological sample from an HCV-infected individual.
In certain embodiments, the conformational epitope comprises the
amino acid sequence depicted in FIGS. 3A 3D, or an amino acid
sequence with at least 80% sequence identity thereto, or 90%
sequence identity thereto, or at least 98% sequence identity
thereto, or any integer in between, so long as the sequence has
protease activity; and the multiple epitope fusion antigen
comprises the amino acid sequence depicted in FIGS. 5A 5F, or an
amino acid sequence with at least 80% sequence identity thereto, or
90% sequence identity thereto, or at least 98% sequence identity
thereto, or any integer in between, so long as the sequence reacts
specifically with anti-HCV antibodies present in a biological
sample from an HCV-infected individual.
In still further embodiments, the NS3/4a conformational epitope
consists of the amino acid sequence depicted in FIGS. 3A 3D and the
multiple epitope fusion antigen consists of the amino acid sequence
depicted in FIGS. 5A 5F.
In another embodiment, the invention is directed to a method of
producing an immunoassay solid support, comprising:
(a) providing a solid support; and
(b) binding to the solid support at least one HCV NS3/4a
conformational epitope and a multiple epitope fusion antigen,
wherein said NS3/4a conformational epitope consists of the amino
acid sequence depicted in FIGS. 3A 3D, and said multiple epitope
fusion antigen consists of the amino acid sequence depicted in
FIGS. 5A 5F.
In still a further embodiment, the subject invention is directed to
a multiple epitope fusion antigen comprising the amino acid
sequence depicted in FIGS. 5A 5F, or an amino acid sequence with at
least 80% sequence identity thereto, or 90% sequence identity
thereto, or an amino acid sequence with at least 98% sequence
identity thereto, or any integer in between, which sequence reacts
specifically with anti-HCV antibodies present in a biological
sample from an HCV-infected individual.
In certain embodiments, the multiple epitope fusion antigen
consists of the amino acid sequence depicted in FIGS. 5A 5F.
In other embodiments, the invention is directed to a polynucleotide
comprising a coding sequence for the multiple epitope fusion
antigen, a recombinant vector comprising the polynucleotide and
control elements operably linked to said polynucleotide whereby the
coding sequence can be transcribed and translated in a host cell, a
host cell transformed with the recombinant vector, and a method of
producing a recombinant multiple epitope fusion antigen comprising
providing a population of host cells as above and culturing said
population of cells under conditions whereby the multiple epitope
fusion antigen encoded by the coding sequence present in said
recombinant vector is expressed.
These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings. In addition, various references are set forth
herein which describe in more detail certain procedures or
compositions, and are therefore incorporated by reference in their
entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagrammatic representation of the HCV genome,
depicting the various regions of the polyprotein from which the
present assay reagents (proteins and antibodies) are derived.
FIG. 2 is a schematic drawing of a representative immunoassay under
the invention.
FIGS. 3A through 3D depict the DNA (SEC ID NO:1) and corresponding
amino acid (SEC ID NO:2) sequence of a representative NS3/4a
conformational antigen for use in the present assays. The amino
acids at positions 403 and 404 of FIGS. 3A through 3D represent
substitutions of Pro for Thr, and Ile for Ser, of the native amino
acid sequence of HCV-1.
FIG. 4 is a diagrammatic representation of MEFA 7.1.
FIGS. 5A 5F depict the DNA (SEC ID NO:3) and corresponding amino
acid (SEC ID NO:4) sequence of MEFA 7.1.
FIGS. 6A 6C show representative MEFAs for use with the subject
immunoassays. FIG. 6A is a diagrammatic representation of MEFA 3.
FIG. 6B is a diagrammatic representation of MEFA 5. FIG. 6C is a
diagrammatic representation of MEFA 6.
FIGS. 7A 7D are diagrams of the construction of psMEFA7.
FIG. 8 is a diagram of the construction of psMEFA7.1.
FIG. 9 is a diagram of the construction of pd.HCV1a.ns3ns4aPI.
DETAILED DESCRIPTION OF THE INVENTION
The practice of the present invention will employ, unless otherwise
indicated, conventional methods of chemistry, biochemistry,
recombinant DNA techniques and immunology, within the skill of the
art. Such techniques are explained fully in the literature. See,
e.g., Fundamental Virology, 2nd Edition, vol. I & II (B. N.
Fields and D. M. Knipe, eds.); Handbook of Experimental Immunology,
Vols. I IV (D. M. Weir and C. C. Blackwell eds., Blackwell
Scientific Publications); T. E. Creighton, Proteins: Structures and
Molecular Properties (W.H. Freeman and Company, 1993); A. L.
Lehninger, Biochemistry (Worth Publishers, Inc., current addition);
Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd
Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan
eds., Academic Press, Inc.).
All publications, patents and patent applications cited herein,
whether supra or infra, are hereby incorporated by reference in
their entirety.
It must be noted that, as used in this specification and the
appended claims, the singular forms. "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
Thus, for example, reference to "an antigen" includes a mixture of
two or more antigens, and the like.
The following amino acid abbreviations are used throughout the
text:
TABLE-US-00001 Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn
(N) Aspartic acid: Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q)
Glutamic acid: Glu (E) Glycine: Gly (G) Histidine: His (H)
Isoleucine: Ile (I) Leucine: Leu (L) Lysine: Lys (K) Methionine:
Met (M) Phenylalanine: Phe (F) Proline: Pro (P) Serine: Ser (S)
Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr (Y) Valine:
Val (V)
I. Definitions
In describing the present invention, the following terms will be
employed, and are intended to be defined as indicated below.
The terms "polypeptide" and "protein" refer to a polymer of amino
acid residues and are not limited to a minimum length of the
product. Thus, peptides, oligopeptides, dimers, multimers, and the
like, are included within the definition. Both full-length proteins
and fragments thereof are encompassed by the definition. The terms
also include postexpression modifications of the polypeptide, for
example, glycosylation, acetylation, phosphorylation and the like.
Furthermore, for purposes of the present invention, a "polypeptide"
refers to a protein which includes modifications, such as
deletions, additions and substitutions (generally conservative in
nature), to the native sequence, so long as the protein maintains
the desired activity. These modifications may be deliberate, as
through site-directed mutagenesis, or may be accidental, such as
through mutations of hosts which produce the proteins or errors due
to PCR amplification.
An HCV polypeptide is a polypeptide, as defined above, derived from
the HCV polyprotein. The polypeptide need not be physically derived
from HCV, but may be synthetically or recombinantly produced.
Moreover, the polypeptide may be derived from any of the various
HCV strains and isolates, such as, but not limited to, any of the
isolates from strains 1, 2, 3, 4, 5 or 6 of HCV. A number of
conserved and variable regions are known between these strains and,
in general, the amino acid sequences of epitopes derived from these
regions will have a high degree of sequence homology, e.g., amino
acid sequence homology of more than 30%, preferably more than 40%,
when the two sequences are aligned. Thus, for example, the term
"NS3/4a" polypeptide refers to native NS3/4a from any of the
various HCV strains, as well as NS3/4a analogs, muteins and
immunogenic fragments, as defined further below. The complete
genotypes of many of these strains are known. See, e.g., U.S. Pat.
No. 6,150,087 and GenBank Accession Nos. AJ238800 and AJ238799.
The terms "analog" and "mutein" refer to biologically active
derivatives of the reference molecule, or fragments of such
derivatives, that retain desired activity, such as immunoreactivity
in the assays described herein. In general, the term "analog"
refers to compounds having a native polypeptide sequence and
structure with one or more amino acid additions, substitutions
(generally conservative in nature) and/or deletions, relative to
the native molecule, so long as the modifications do not destroy
immunogenic activity. The term "mutein" refers to peptides having
one or more peptide mimics ("peptoids"), such as those described in
International Publication No. WO 91/04282. Preferably, the analog
or mutein has at least the same immunoactivity as the native
molecule. Methods for making polypeptide analogs and muteins are
known in the art and are described further below.
Particularly preferred analogs include substitutions that are
conservative in nature, i.e., those substitutions that take place
within a family of amino acids that are related in their side
chains. Specifically, amino acids are generally divided into four
families: (1) acidic--aspartate and glutamate; (2) basic--lysine,
arginine, histidine; (3) non-polar--alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan; and (4)
uncharged polar--glycine, asparagine, glutamine, cysteine, serine
threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are
sometimes classified as aromatic amino acids. For example, it is
reasonably predictable that an isolated replacement of leucine with
isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar conservative replacement of an amino
acid with a structurally related amino acid, will not have a major
effect on the biological activity. For example, the polypeptide of
interest may include up to about 5 10 conservative or
non-conservative amino acid substitutions, or even up to about 15
25 conservative or non-conservative amino acid substitutions, or
any integer between 5 25, so long as the desired function of the
molecule remains intact. One of skill in the art may readily
determine regions of the molecule of interest that can tolerate
change by reference to Hopp/Woods and Kyte-Doolittle plots, well
known in the art.
By "fragment" is intended a polypeptide consisting of only a part
of the intact full-length polypeptide sequence and structure. The
fragment can include a C-terminal deletion and/or an N-terminal
deletion of the native polypeptide. An "immunogenic fragment" of a
particular HCV protein will generally include at least about 5 10
contiguous amino acid residues of the full-length molecule,
preferably at least about 15 25 contiguous amino acid residues of
the full-length molecule, and most preferably at least about 20 50
or more contiguous amino acid residues of the full-length molecule,
that define an epitope, or any integer between 5 amino acids and
the full-length sequence, provided that the fragment in question
retains immunoreactivity in the assays described herein. For
example, preferred immunogenic fragments, include but are not
limited to fragments of HCV core that comprise, e.g., amino acids
10 45, 10 53, 67 88, and 120 130 of the polyprotein, epitope 5-1-1
(in the NS4a/NS4b region of the viral genome) as well as defined
epitopes derived from any of the regions of the polyprotein shown
in FIG. 1, such as but not limited to the E1, E2, NS3 (e.g.,
polypeptide c33c from the NS3 region), NS4 (e.g., polypeptide c100
from the NS3/NS4 regions), NS3/4a and NS5 regions of the HCV
polyprotein, as well as any of the other various epitopes
identified from the HCV polyprotein. See, e.g., Chien et al., Proc.
Natl. Acad. Sci. USA (1992) 89:10011 10015; Chien et al., J.
Gastroent. Hepatol. (1993) 8:S33 39; Chien et al., International
Publication No. WO 93/00365; Chien, D. Y., International
Publication No. WO 94/01778; U.S. Pat. Nos. 6,150,087 and
6,121,020, all of which are incorporated by reference herein in
their entireties.
The term "epitope" as used herein refers to a sequence of at least
about 3 to 5, preferably about 5 to 10 or 15, and not more than
about 1,000 amino acids (or any integer therebetween), which define
a sequence that by itself or as part of a larger sequence, binds to
an antibody generated in response to such sequence. There is no
critical upper limit to the length of the fragment, which may
comprise nearly the full-length of the protein sequence, or even a
fusion protein comprising two or more epitopes from the HCV
polyprotein. An epitope for use in the subject invention is not
limited to a polypeptide having the exact sequence of the portion
of the parent protein from which it is derived. Indeed, viral
genomes are in a state of constant flux and contain several
variable domains which exhibit relatively high degrees of
variability between isolates. Thus the term "epitope" encompasses
sequences identical to the native sequence, as well as
modifications to the native sequence, such as deletions, additions
and substitutions (generally conservative in nature).
Regions of a given polypeptide that include an epitope can be
identified using any number of epitope mapping techniques, well
known in the art. See, e.g., Epitope Mapping Protocols in Methods
in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana
Press, Totowa, N.J. For example, linear epitopes may be determined
by e.g., concurrently synthesizing large numbers of peptides on
solid supports, the peptides corresponding to portions of the
protein molecule, and reacting the peptides with antibodies while
the peptides are still attached to the supports. Such techniques
are known in the art and described in, e.g., U.S. Pat. No.
4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998
4002; Geysen et al. (1985) Proc. Natl. Acad. Sci. USA 82:178 182;
Geysen et al. (1986) Molec. Immunol. 23:709 715, all incorporated
herein by reference in their entireties. Using such techniques, a
number of epitopes of HCV have been identified. See, e.g., Chien et
al., Viral Hepatitis and Liver Disease (1994) pp. 320 324, and
further below. Similarly, conformational epitopes are readily
identified by determining spatial conformation of amino acids such
as by, e.g., x-ray crystallography and 2-dimensional nuclear
magnetic resonance. See, e.g., Epitope Mapping Protocols, supra.
Antigenic regions of proteins can also be identified using standard
antigenicity and hydropathy plots, such as those calculated using,
e.g., the Omiga version 1.0 software program available from the
Oxford Molecular Group. This computer program employs the
Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981)
78:3824 3828 for determining antigenicity profiles, and the
Kyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105
132 for hydropathy plots.
As used herein, the term "conformational epitope" refers to a
portion of a full-length protein, or an analog or mutein thereof,
having structural features native to the amino acid sequence
encoding the epitope within the full-length natural protein. Native
structural features include, but are not limited to, glycosylation
and three dimensional structure. The length of the epitope defining
sequence can be subject to wide variations as these epitopes are
believed to be formed by the three-dimensional shape of the antigen
(e.g., folding). Thus, amino acids defining the epitope can be
relatively few in number, but widely dispersed along the length of
the molecule, being brought into correct epitope conformation via
folding. The portions of the antigen between the residues defining
the epitope may not be critical to the conformational structure of
the epitope. For example, deletion or substitution of these
intervening sequences may not affect the conformational epitope
provided sequences critical to epitope conformation are maintained
(e.g., cysteines involved in disulfide bonding, glycosylation
sites, etc.).
Conformational epitopes present in the NS3/4a region are readily
identified using methods discussed above. Moreover, the presence or
absence of a conformational epitope in a given polypeptide can be
readily determined through screening the antigen of interest with
an antibody (polyclonal serum or monoclonal to the conformational
epitope) and comparing its reactivity to that of a denatured
version of the antigen which retains only linear epitopes (if any).
In such screening using polyclonal antibodies, it may be
advantageous to absorb the polyclonal serum first with the
denatured antigen and see if it retains antibodies to the antigen
of interest. Additionally, in the case of NS3/4a, a molecule which
preserves the native conformation will also have protease and,
optionally, helicase enzymatic activities. Such activities can be
detected using enzymatic assays, as described further below.
Preferably, a conformational epitope is produced recombinantly and
is expressed in a cell from which it is extractable under
conditions which preserve its desired structural features, e.g.
without denaturation of the epitope. Such cells include bacteria,
yeast, insect, and mammalian cells. Expression and isolation of
recombinant conformational epitopes from the HCV polyprotein are
described in e.g., International Publication Nos. WO 96/04301, WO
94/01778, WO 95/33053, WO 92/08734, which applications are herein
incorporated by reference in their entirety. Alternatively, it is
possible to express the antigens and further renature the protein
after recovery. It is also understood that chemical synthesis may
also provide conformational antigen mimitopes that cross-react with
the "native" antigen's conformational epitope.
The term "multiple epitope fusion antigen" or "MEFA" as used herein
intends a polypeptide in which multiple HCV antigens are part of a
single, continuous chain of amino acids, which chain does not occur
in nature. The HCV antigens may be connected directly to each other
by peptide bonds or may be separated by intervening amino acid
sequences. The fusion antigens may also contain sequences exogenous
to the HCV polyprotein. Moreover, the HCV sequences present may be
from multiple genotypes and/or isolates of HCV. Examples of
particular MEFAs for use in the present immunoassays are detailed
in, e.g., International Publication No. WO 97/44469, incorporated
herein by reference in its entirety, and are described further
below.
An "antibody" intends a molecule that, through chemical or physical
means, specifically binds to a polypeptide of interest. Thus, for
example, an HCV core antibody is a molecule that specifically binds
to the HCV core protein. The term "antibody" as used herein
includes antibodies obtained from both polyclonal and monoclonal
preparations, as well as, the following: hybrid (chimeric) antibody
molecules (see, for example, Winter et al. (1991) Nature 349:293
299; and U.S. Pat. No. 4,816,567); F(ab').sub.2 and F(ab)
fragments; Fv molecules (non-covalent heterodimers, see, for
example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659 2662;
and Ehrlich et al. (1980) Biochem 19:4091 4096); single-chain Fv
molecules (sFv) (see, for example, Huston et al. (1988) Proc Natl
Acad Sci USA 85:5879 5883); dimeric and trimeric antibody fragment
constructs; minibodies (see, e.g., Pack et al. (1992) Biochem
31:1579 1584; Cumber et al. (1992) J. Immunology 149B:120 126);
humanized antibody molecules (see, for example, Riechmann et al.
(1988) Nature 332:323 327; Verhoeyan et al. (1988) Science 239:1534
1536; and U.K. Patent Publication No. GB 2,276,169, published 21
Sep. 1994); and, any functional fragments obtained from such
molecules, wherein such fragments retain immunological binding
properties of the parent antibody molecule.
As used herein, the term "monoclonal antibody" refers to an
antibody composition having a homogeneous antibody population. The
term is not limited regarding the species or source of the
antibody, nor is it intended to be limited by the manner in which
it is made. Thus, the term encompasses antibodies obtained from
murine hybridomas, as well as human monoclonal antibodies obtained
using human rather than murine hybridomas. See, e.g., Cote, et al.
Monclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, p.
77.
By "isolated" is meant, when referring to a polypeptide, that the
indicated molecule is separate and discrete from the whole organism
with which the molecule is found in nature or is present in the
substantial absence of other biological macro-molecules of the same
type. The term "isolated" with respect to a polynucleotide is a
nucleic acid molecule devoid, in whole or part, of sequences
normally associated with it in nature; or a sequence, as it exists
in nature, but having heterologous sequences in association
therewith; or a molecule disassociated from the chromosome.
By "equivalent antigenic determinant" is meant an antigenic
determinant from different sub-species or strains of HCV, such as
from strains 1, 2, or 3 of HCV. More specifically, epitopes are
known, such as 5-1-1, and such epitopes vary between the strains 1,
2, and 3. Thus, the epitope 5-1-1 from the three different strains
are equivalent antigenic determinants and thus are "copies" even
though their sequences are not identical. In general the amino acid
sequences of equivalent antigenic determinants will have a high
degree of sequence homology, e.g., amino acid sequence homology of
more than 30%, preferably more than 40%, when the two sequences are
aligned.
"Homology" refers to the percent similarity between two
polynucleotide or two polypeptide moieties. Two DNA, or two
polypeptide sequences are "substantially homologous" to each other
when the sequences exhibit at least about 50% , preferably at least
about 75%, more preferably at least about 80% 85%, preferably at
least about 90%, and most preferably at least about 95% 98%
sequence similarity over a defined length of the molecules. As used
herein, substantially homologous also refers to sequences showing
complete identity to the specified DNA or polypeptide sequence.
In general, "identity" refers to an exact nucleotide-to-nucleotide
or amino acid-to-amino acid correspondence of two polynucleotides
or polypeptide sequences, respectively. Percent identity can be
determined by a direct comparison of the sequence information
between two molecules by aligning the sequences, counting the exact
number of matches between the two aligned sequences, dividing by
the length of the shorter sequence, and multiplying the result by
100.
Readily available computer programs can be used to aid in the
analysis of homology and identity, such as ALIGN, Dayhoff, M. O. in
Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5 Suppl.
3:353 358, National biomedical Research Foundation, Washington, DC,
which adapts the local homology algorithm of Smith and Waterman
Advances in Appl. Math. 2:482 489, 1981 for peptide analysis.
Programs for determining nucleotide sequence homology are available
in the Wisconsin Sequence Analysis Package, Version 8 (available
from Genetics Computer Group, Madison, Wis.) for example, the
BESTFIT, FASTA and GAP programs, which also rely on the Smith and
Waterman algorithm. These programs are readily utilized with the
default parameters recommended by the manufacturer and described in
the Wisconsin Sequence Analysis Package referred to above. For
example, percent homology of a particular nucleotide sequence to a
reference sequence can be determined using the homology algorithm
of Smith and Waterman with a default scoring table and a gap
penalty of six nucleotide positions.
Another method of establishing percent homology in the context of
the present invention is to use the MPSRCH package of programs
copyrighted by the University of Edinburgh, developed by John F.
Collins and Shane S. Sturrok, and distributed by IntelliGenetics,
Inc. (Mountain View, Calif.). From this suite of packages the
Smith-Waterman algorithm can be employed where default parameters
are used for the scoring table (for example, gap open penalty of
12, gap extension penalty of one, and a gap of six). From the data
generated the "Match" value reflects "sequence homology." Other
suitable programs for calculating the percent identity or
similarity between sequences are generally known in the art, for
example, another alignment program is BLAST, used with default
parameters. For example, BLASTN and BLASTP can be used using the
following default parameters: genetic code=standard; filter=none;
strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50
sequences; sort by=HIGH SCORE; Databases=non-redundant,
GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss
protein+Spupdate+PIR.
Alternatively, homology can be determined by hybridization of
polynucleotides under conditions which form stable duplexes between
homologous regions, followed by digestion with
single-stranded-specific nuclease(s), and size determination of the
digested fragments. DNA sequences that are substantially homologous
can be identified in a Southern hybridization experiment under, for
example, stringent conditions, as defined for that particular
system. Defining appropriate hybridization conditions is within the
skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning,
supra; Nucleic Acid Hybridization, supra.
A "coding sequence" or a sequence which "encodes" a selected
polypeptide, is a nucleic acid molecule which is transcribed (in
the case of DNA) and translated (in the case of MRNA) into a
polypeptide in vitro or in vivo when placed under the control of
appropriate regulatory sequences. The boundaries of the coding
sequence are determined by a start codon at the 5' (amino) terminus
and a translation stop codon at the 3' (carboxy) terminus. A
transcription termination sequence may be located 3' to the coding
sequence.
"Operably linked" refers to an arrangement of elements wherein the
components so described are configured so as to perform their
desired function. Thus, a given promoter operably linked to a
coding sequence is capable of effecting the expression of the
coding sequence when the proper transcription factors, etc., are
present. The promoter need not be contiguous with the coding
sequence, so long as it functions to direct the expression thereof.
Thus, for example, intervening untranslated yet transcribed
sequences can be present between the promoter sequence and the
coding sequence, as can transcribed introns, and the promoter
sequence can still be considered "operably linked" to the coding
sequence.
"Recombinant" as used herein to describe a nucleic acid molecule
means a polynucleotide of genomic, cDNA, viral, semisynthetic, or
synthetic origin which, by virtue of its origin or manipulation is
not associated with all or a portion of the polynucleotide with
which it is associated in nature. The term "recombinant" as used
with respect to a protein or polypeptide means a polypeptide
produced by expression of a recombinant polynucleotide. In general,
the gene of interest is cloned and then expressed in transformed
organisms, as described further below. The host organism expresses
the foreign gene to produce the protein under expression
conditions.
A "control element" refers to a polynucleotide sequence which aids
in the expression of a coding sequence to which it is linked. The
term includes promoters, transcription termination sequences,
upstream regulatory domains, polyadenylation signals, untranslated
regions, including 5'-UTRs and 3'-UTRs and when appropriate, leader
sequences and enhancers, which collectively provide for the
transcription and translation of a coding sequence in a host
cell.
A "promoter" as used herein is a DNA regulatory region capable of
binding RNA polymerase in a host cell and initiating transcription
of a downstream (3' direction) coding sequence operably linked
thereto. For purposes of the present invention, a promoter sequence
includes the minimum number of bases or elements necessary to
initiate transcription of a gene of interest at levels detectable
above background. Within the promoter sequence is a transcription
initiation site, as well as protein binding domains (consensus
sequences) responsible for the binding of RNA polymerase.
Eucaryotic promoters will often, but not always, contain "TATA"
boxes and "CAT" boxes.
A control sequence "directs the transcription" of a coding sequence
in a cell when RNA polymerase will bind the promoter sequence and
transcribe the coding sequence into mRNA, which is then translated
into the polypeptide encoded by the coding sequence.
"Expression cassette" or "expression construct" refers to an
assembly which is capable of directing the expression of the
sequence(s) or gene(s) of interest. The expression cassette
includes control elements, as described above, such as a promoter
which is operably linked to (so as to direct transcription of) the
sequence(s) or gene(s) of interest, and often includes a
polyadenylation sequence as well. Within certain embodiments of the
invention, the expression cassette described herein may be
contained within a plasmid construct. In addition to the components
of the expression cassette, the plasmid construct may also include,
one or more selectable markers, a signal which allows the plasmid
construct to exist as single-stranded DNA (e.g., a M13 origin of
replication), at least one multiple cloning site, and a "mammalian"
origin of replication (e.g., a SV40 or adenovirus origin of
replication).
"Transformation," as used herein, refers to the insertion of an
exogenous polynucleotide into a host cell, irrespective of the
method used for insertion: for example, transformation by direct
uptake, transfection, infection, and the like. For particular
methods of transfection, see further below. The exogenous
polynucleotide may be maintained as a nonintegrated vector, for
example, an episome, or alternatively, may be integrated into the
host genome.
A "host cell" is a cell which has been transformed, or is capable
of transformation, by an exogenous DNA sequence.
As used herein, a "biological sample" refers to a sample of tissue
or fluid isolated from a subject, that commonly includes antibodies
produced by the subject. Typical samples that include such
antibodies are known in the art and include but not limited to,
blood, plasma, serum, fecal matter, urine, bone marrow, bile,
spinal fluid, lymph fluid, samples of the skin, secretions of the
skin, respiratory, intestinal, and genitourinary tracts, tears,
saliva, milk, blood cells, organs, biopsies and also samples of in
vitro cell culture constituents including but not limited to
conditioned media resulting from the growth of cells and tissues in
culture medium, e.g., recombinant cells, and cell components.
"Common solid support" intends a single solid matrix to which the
HCV polypeptides used in the subject immunoassays are bound
covalently or by noncovalent means such as hydrophobic
adsorption.
"Immunologically reactive" means that the antigen in question will
react specifically with anti-HCV antibodies present in a biological
sample from an HCV-infected individual.
"Immune complex" intends the combination formed when an antibody
binds to an epitope on an antigen.
As used herein, the terms "label" and "detectable label" refer to a
molecule capable of detection, including, but not limited to,
radioactive isotopes, fluorescers, chemiluminescers, chromophores,
enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,
chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin,
avidin, strepavidin or haptens) and the like. The term "fluorescer"
refers to a substance or a portion thereof which is capable of
exhibiting fluorescence in the detectable range. Particular
examples of labels which may be used under the invention include,
but are not limited to, horse radish peroxidase (HRP), fluorescein,
FITC, rhodamine, dansyl, umbelliferone, dimethyl acridinium ester
(DMAE), Texas red, luminol, NADPH and
.alpha.-.beta.-galactosidase.
II. Modes of Carrying out the Invention
Before describing the present invention in detail, it is to be
understood that this invention is not limited to particular
formulations or process parameters as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments of the invention
only, and is not intended to be limiting.
Although a number of compositions and methods similar or equivalent
to those described herein can be used in the practice of the
present invention, the preferred materials and methods are
described herein.
As noted above, the present invention is based on the discovery of
novel diagnostic methods for accurately detecting early HCV
infection. The methods rely on the identification and use of highly
immunogenic HCV antigens which are present during the early stages
of HCV seroconversion, thereby increasing detection accuracy and
reducing the incidence of false results. In particular, the
immunoassays described herein utilize highly immunogenic
conformational epitopes derived from the NS3/4a region of the HCV
polyprotein, and multiple epitope fusion antigens comprising
various HCV polypeptides, either from the same or different HCV
genotypes and isolates, such as multiple immunodominant epitopes,
for example, major linear epitopes of HCV core, E1, E2, NS3, 5-1-1,
c100-3 and NS5 sequences. The methods can be conveniently practiced
in a single assay, using any of the several assay formats described
below, such as but not limited to, assay formats which utilize a
solid support to which the HCV antigens are bound.
The NS3/4a region of the HCV polyprotein has been described and the
amino acid sequence and overall structure of the protein are
disclosed in, e.g., Yao et al., Structure (November 1999) 7:1353
1363; Sali et al., Biochem. (1998) 37:3392 3401; and
Bartenschlager, R., J. Viral Hepat. (1999) 6:165 181. See, also,
Dasmahapatra et al., U.S. Pat. No. 5,843,752, incorporated herein
by reference in its entirety. The subject immunoassays utilize at
least one conformational epitope derived from the NS3/4a region
that exists in the conformation as found in the naturally occurring
HCV particle or its infective product, as evidenced by the
preservation of protease and, optionally, helicase enzymatic
activities normally displayed by the NS3/4a gene product and/or
immunoreactivity of the antigen with antibodies in a biological
sample from an HCV-infected subject, and a loss of the epitope's
immunoreactivity upon denaturation of the antigen. For example, the
conformational epitope can be disrupted by heating, changing the pH
to extremely acid or basic, or by adding known organic denaturants,
such as dithiothreitol (DTT) or an appropriate detergent. See,
e.g., Protein Purification Methods, a practical approach (E. L. V.
Harris and S. Angal eds., IRL Press) and the denatured product
compared to the product which is not treated as above.
Protease and helicase activity may be determined using standard
enzyme assays well known in the art. For example, protease activity
may be determined using the procedure described below in the
examples, as well as using assays well known in the art. See, e.g.,
Takeshita et al., Anal Biochem. (1997) 247:242 246; Kakiuchi et
al., J. Biochem. (1997) 122:749 755; Sali et al., Biochemistry
(1998) 37:3392 3401; Cho et al., J. Virol. Meth. (1998) 72:109 115;
Cerretani et al., Anal. Biochem. (1999) 266:192 197; Zhang et al.,
Anal. Biochem. (1999) 270:268 275; Kakiuchi et al., J. Virol. Meth.
(1999) 80:77 84; Fowler et al., J. Biomol. Screen. (2000) 5:153
158; and Kim et al., Anal. Biochem. (2000) 284:42 48. Similarly,
helicase activity assays are well known in the art and helicase
activity of an NS3/4a epitope may be determined using, for example,
an ELISA assay, as described in, e.g., Hsu et al., Biochem.
Biophys. Res. Commun. (1998) 253:594 599; a scintillation proximity
assay system, as described in Kyono et al., Anal. Biochem. (1998)
257:120 126; high throughput screening assays as described in,
e.g., Hicham et al., Antiviral Res. (2000) 46:181 193 and Kwong et
al., Methods Mol. Med. (2000) 24:97 116; as well as by other assay
methods known in the art. See, e.g., Khu et al., J. Virol. (2001)
75:205 214; Utama et al., Virology (2000) 273:316 324; Paolini et
al., J. Gen. Virol. (2000) 81:1335 1345; Preugschat et al.,
Biochemistry (2000) 39:5174 5183; Preugschat et al., Methods Mol.
Med. (1998) 19:353 364; and Hesson et al., Biochemistry (2000)
39:2619 2625.
The length of the antigen is sufficient to maintain an
immunoreactive conformational epitope. Often, the polypeptide
containing the antigen used will be almost full-length, however,
the polypeptide may also be truncated to, for example, increase
solubility or to improve secretion. Generally, the conformational
epitope found in NS3/4a is expressed as a recombinant polypeptide
in a cell and this polypeptide provides the epitope in a desired
form, as described in detail below.
A representative amino acid sequence for the NS3/4a polypeptide is
shown in FIGS. 3A through 3D. The amino acid sequence shown at
positions 2 686 of the figure corresponds to amino acid positions
1027 1711 of HCV-1. An initiator codon (ATG) coding for Met, is
shown as position 1. Additionally, the Thr normally occurring at
position 1428 of HCV-1 (amino acid position 403 of FIG. 3) is
mutated to Pro, and the Ser normally occurring at position 1429 of
HCV-1 (amino acid position 404 of FIG. 3) is mutated to Ile.
However, either the native sequence, with or without an N-terminal
Met, the depicted analog, with or without the N-terminal Met, or
other analogs and fragments can be used in the subject assays, so
long as the epitope is produced using a method that retains or
reinstates its native conformation such that protease activity, and
optionally, helicase activity is retained. Dasmahapatra et al.,
U.S. Pat. No. 5,843,752 and Zhang et al., U.S. Pat. No. 5,990,276,
both describe analogs of NS3/4a.
The NS3 protease of NS3/4a is found at about positions 1027 1207,
numbered relative to HCV-1 (see, Choo et al., Proc. Natl. Acad.
Sci. USA (1991) 88:2451 2455), positions 2 182 of FIG. 3. The
structure of the NS3 protease and active site are known. See, e.g.,
De Francesco et al., Antivir. Ther. (1998) 3:99 109; Koch et al.,
Biochemistry (2001) 40:631 640. Changes to the native sequence that
will normally be tolerated will be those outside of the active site
of the molecule. Particularly, it is desirable to maintain amino
acids 1- or 2 155 of FIG. 3, with little or only conservative
substitutions. Amino acids occurring beyond 155 will tolerate
greater changes. Additionally, if fragments of the NS3/4a sequence
found in FIG. 3 are used, these fragments will generally include at
least amino acids 1- or 2 155, preferably amino acids 1- or 2 175,
and most preferably amino acids 1- or 2 182, with or without the
N-terminal Met. The helicase domain is found at about positions
1193 1657 of HCV-1 (positions 207 632 of FIG. 3). Thus, if helicase
activity is desired, this portion of the molecule will be
maintained with little or only conservative changes. One of skill
in the art can readily determine other regions that will tolerate
change based on the known structure of NS3/4a.
The immunoassays described herein also utilize multiple epitope
fusion antigens (termed "MEFAs"), as described in International
Publication No. WO 97/44469. Such MEFAs include multiple epitopes
derived from two or more of the various viral regions of the HCV
polyprotein shown in FIG. 1 and Table 1. In particular, as shown in
FIG. 1 and Table 1, An HCV polyprotein, upon cleavage, produces at
least ten distinct products, in the order of NH.sub.2-
Core-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a- NS5b-COOH. The core
polypeptide occurs at positions 1 191, numbered relative to HCV-1
(see, Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451 2455,
for the HCV-1 genome). This polypeptide is further processed to
produce an HCV polypeptide with approximately amino acids 1 173.
The envelope polypeptides, E1 and E2, occur at about positions 192
383 and 384 746, respectively. The P7 domain is found at about
positions 747 809. NS2 is an integral membrane protein with
proteolytic activity and is found at about positions 810 1026 of
the polyprotein. NS2, either alone or in combination with NS3
(found at about positions 1027 1657), cleaves the NS2 NS3 sissle
bond which in turn generates the NS3 N-terminus and releases a
large polyprotein that includes both serine protease and RNA
helicase activities. The NS3 protease, found at about positions
1027 1207, serves to process the remaining polyprotein. The
helicase activity is found at about positions 1193 1657. Completion
of polyprotein maturation is initiated by autocatalytic cleavage at
the NS3 NS4a junction, catalyzed by the NS3 serine protease.
Subsequent NS3-mediated cleavages of the HCV polyprotein appear to
involve recognition of polyprotein cleavage junctions by an NS3
molecule of another polypeptide. In these reactions, NS3 liberates
an NS3 cofactor (NS4a, found about positions 1658 1711), two
proteins (NS4b found at about positions 1712 1972, and NS5a found
at about positions 1973 2420), and an RNA-dependent RNA polymerase
(NS5b found at about positions 2421 3011).
TABLE-US-00002 TABLE 1 Domain Approximate Boundaries* C (core) 1
191 E1 192 383 E2 384 746 P7 747 809 NS2 810 1026 NS3 1027 1657
NS4a 1658 1711 NS4b 1712 1972 NS5a 1973 2420 NS5b 2421 3011
*Numbered relative to HCV-1. See, Choo et al. (1991) Proc. Natl.
Acad. Sci. USA 88: 2451 2455.
The multiple HCV antigens are part of a single, continuous chain of
amino acids, which chain does not occur in nature. Thus, the linear
order of the epitopes is different than their linear order in the
genome in which they occur. The linear order of the sequences of
the MEFAs for use herein is preferably arranged for optimum
antigenicity. Preferably, the epitopes are from more than one HCV
strain, thus providing the added ability to detect multiple strains
of HCV in a single assay. Thus, the MEFAs for use herein may
comprise various immunogenic regions derived from the polyprotein
described above. Moreover, a protein resulting from a frameshift in
the core region of the polyprotein, such as described in
International Publication No. WO 99/63941, may be used in the
MEFAs. If desired, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more
of one or more epitopes derived from the HCV polyprotein may occur
in the fusion protein.
For example, epitopes derived from, e.g., the hypervariable region
of E2, such as a region spanning amino acids 384 410 or 390 410,
can be included in the MEFA antigen. A particularly effective E2
epitope is one which includes a consensus sequence derived from
this region, such as the consensus sequence
Gly-Ser-Ala-Ala-Arg-Thr-Thr-Ser-Gly-Phe-Val-Ser-Leu-Phe-Ala-Pro-Gly-Ala-L-
ys-Gln-Asn (SEC ID NO:5), which represents a consensus sequence for
amino acids 390 410 of the HCV type 1 genome. A representative E2
epitope present in a MEFA of the invention can comprise a hybrid
epitope spanning amino acids 390 444. Such a hybrid E2 epitope can
include a consensus sequence representing amino acids 390 410 fused
to the native amino acid sequence for amino acids 411 444 of HCV
E2.
Additionally, the antigens may be derived from various HCV strains.
Multiple viral strains of HCV are known, and epitopes derived from
any of these strains can be used in a fusion protein. It is well
known that any given species of organism varies from one individual
organism to another and further that a given organism such as a
virus can have a number of different strains. For example, as
explained above, HCV includes at least 6 genotypes. Each of these
genotypes includes equivalent antigenic determinants. More
specifically, each strain includes a number of antigenic
determinants that are present on all strains of the virus but are
slightly different from one viral strain to another. For example,
HCV includes the antigenic determinant known as 5-1-1 (See, FIG.
1). This particular antigenic determinant appears in three
different forms on the three different viral strains of HCV.
Accordingly, in a preferred embodiment of the invention all three
forms of 5-1-1 appear on the multiple epitope fusion antigen used
in the subject immunoassays. Similarly, equivalent antigenic
determinants from the core region of different HCV strains may also
be present. In general, equivalent antigenic determinants have a
high degree of homology in terms of amino acid sequence which
degree of homology is generally 30% or more, preferably 40% or
more, when aligned. The multiple copy epitope of the present
invention can also include multiple copies which are exact copies
of the same epitope.
FIGS. 4 and 6A 6C show representative MEFAs for use in the present
invention which are derived from HCV. However, it is to be
understood that other epitopes derived from the HCV genome will
also find use with the present assays.
The DNA sequence and corresponding amino acid sequence of a
representative multiple epitope fusion antigen, MEFA 7.1, is shown
in FIGS. 5A through 5F. The general structural formula for MEFA 7.1
is shown in FIG. 4 and is as follows: hSOD-E1(type 1)-E2 HVR
consensus(type 1a)-E2 HVR consensus(types 1 and 2)-helicase(type
1)-5-1-1(type 1)-5-1-1(type 3)-5-1-1(type 2)-c100(type 1)-NS5(type
1)-NS5(type 1)-core(types 1+2)-core(types 1+2). This multiple copy
epitope includes the following amino acid sequence, numbered
relative to HCV-1 (the numbering of the amino acids set forth below
follows the numbering designation provided in Choo, et al. (1991)
Proc. Natl. Acad. Sci. USA 88:2451 2455, in which amino acid #1 is
the first methionine encoded by the coding sequence of the core
region): amino acids 1 156 of superoxide dismutase (SOD, used to
enhance recombinant expression of the protein); amino acids 303 to
320 of the polyprotein from the E1 region; amino acids 390 to 410
of the polyprotein, representing a consensus sequence for the
hypervariable region of HCV-1a E2; amino acids 384 to 414 of the
polyprotein from region E2, representing a consensus sequence for
the E2 hypervariable regions of HCV-1 and HCV-2; amino acids 1193
1658 of the HCV-1 polyprotein which define the helicase; three
copies of an epitope from 5-1-1, amino acids 1689 1735, one from
HCV-1, one from HCV-3 and one from HCV-2, which copies are
equivalent antigenic determinants from the three different viral
strains of HCV; HCV polypeptide C100 of HCV-1, amino acids 1901
1936 of the polyprotein; two exact copies of an epitope from the
NS5 region of HCV-1, each with amino acids 2278 to 2313 of the HCV
polyprotein; and two copies of an epitope from the core region, one
from HCV-1 and one from HCV-2, which copies are equivalent
antigenic determinants represented by amino acids 9 to 32, 39 42
and 64 88 of HCV-1 and 67 84 of HCV-2.
Table 2 shows the amino acid positions of the various epitopes with
reference to FIGS. 5A through 5F herein.
TABLE-US-00003 TABLE 2 MEFA 7.1 mefa aa# 5' end site epitope hcv
aa# strain 1 156 Nco1 hSOD 159 176 EcoR1 E1 303 320 1 179 199
Hind111 E2 HVR1a 390 410 1 consensus 200 230 E2 HLVR1 + 2 384 414 1
+ 2 consensus 231 696 Sal1 Helicase 1193 1658 1 699 745 Sph1 5-1-1
1689 1735 1 748 794 Nru1 5-1-1 1689 1735 3 797 843 Cla1 5-1-1 1689
1735 2 846 881 Ava1 C100 1901 1936 1 884 919 Xba1 NS5 2278 2313 1
922 957 Bgl11 NS5 2278 2313 1 958 1028 Nco1 core 9 32, 39 42 1
epitopes 64 88 1 67 84 2 1029 1099 Bal1 core 9 32, 39 42, 1
epitopes 64 88 1 67 84 2
In one embodiment of the invention, depicted in FIG. 2, a rapid
capture ligand immunoassay is performed using a conformational
epitope from NS3/4a and one or more multiple epitope fusion
antigens, such as MEFA 7.1. The sample is combined with the
antigens, which may be present on a solid support, as described
further below. If the sample is infected with HCV, HCV antibodies
to those epitopes present on the solid support will bind to the
solid support components. Detection is by the attachment of a
detectable marker (such as horse radish peroxidase (HRP) as shown
in FIG. 2) to a member of the antigen/antibody complex. Attachment
may be by covalent means or by subsequent binding of detectably
labeled antibodies, such as in a standard sandwich assay, or by
enzyme reaction, the product of which reaction is detectable. The
detectable marker may include, but is not limited to, a
chromophore, an antibody, an antigen, an enzyme, an enzyme reactive
compound whose cleavage product is detectable, rhodamine or
rhodamine derivative, biotin, avidin, strepavidin, a fluorescent
compound, a chemiluminescent compound, such as dimethyl acridinium
ester (DMAE, Ciba Corning Diagnostics Corp.), derivatives and/or
combinations of these markers. A detectably labeled anti-human
antibody, capable of detecting a human IgG molecule present, can be
conveniently used.
In order to further an understanding of the invention, a more
detailed discussion is provided below regarding production of
polypeptides for use in the immunoassays and methods of conducting
the immunoassays.
Production of Antigens for use in the HCV Immunoassays
As explained above, the molecules of the present invention are
generally produced recombinantly. Thus, polynucleotides encoding
HCV antigens for use with the present invention can be made using
standard techniques of molecular biology. For example,
polynucleotide sequences coding for the above-described molecules
can be obtained using recombinant methods, such as by screening
cDNA and genomic libraries from cells expressing the gene, or by
deriving the gene from a vector known to include the same.
Furthermore, the desired gene can be isolated directly from viral
nucleic acid molecules, using techniques described in the art, such
as in Houghton et al., U.S. Pat. No. 5,350,671. The gene of
interest can also be produced synthetically, rather than cloned.
The molecules can be designed with appropriate codons for the
particular sequence. The complete sequence is then assembled from
overlapping oligonucleotides prepared by standard methods and
assembled into a complete coding sequence. See, e.g., Edge (1981)
Nature 292:756; Nambair et al. (1984) Science 223:1299; and Jay et
al. (1984) J. Biol. Chem. 259:6311.
Thus, particular nucleotide sequences can be obtained from vectors
harboring the desired sequences or synthesized completely or in
part using various oligonucleotide synthesis techniques known in
the art, such as site-directed mutagenesis and polymerase chain
reaction (PCR) techniques where appropriate. See, e.g., Sambrook,
supra. In particular, one method of obtaining nucleotide sequences
encoding the desired sequences is by annealing complementary sets
of overlapping synthetic oligonucleotides produced in a
conventional, automated polynucleotide synthesizer, followed by
ligation with an appropriate DNA ligase and amplification of the
ligated nucleotide sequence. via PVR. See, e.g., Jayaraman et al.
(1991) Proc. Natl. Acad. Sci. USA 88:4084 4088. Additionally,
oligonucleotide directed synthesis (Jones et al. (1986) Nature
54:75 82), oligonucleotide directed mutagenesis of pre-existing
nucleotide regions (Riechmann et al. (1988) Nature 332:323 327 and
Verhoeyen et al. (1988) Science 239:1534 1536), and enzymatic
filling-in of gapped oligonucleotides using T.sub.4 DNA polymerase
(Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029 10033) can
be used under the invention to provide molecules having altered or
enhanced antigen-binding capabilities, and/or reduced
immunogenicity.
Once coding sequences have been prepared or isolated, such
sequences can be cloned into any suitable vector or replicon.
Numerous cloning vectors are known to those of skill in the art,
and the selection of an appropriate cloning vector is a matter of
choice. Suitable vectors include, but are not limited to, plasmids,
phages, transposons, cosmids, chromosomes or viruses which are
capable of replication when associated with the proper control
elements.
The coding sequence is then placed under the control of suitable
control elements, depending on the system to be used for
expression. Thus, the coding sequence can be placed under the
control of a promoter, ribosome binding site (for bacterial
expression) and, optionally, an operator, so that the DNA sequence
of interest is transcribed into RNA by a suitable transformant. The
coding sequence may or may not contain a signal peptide or leader
sequence which can later be removed by the host in
post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739;
4,425,437; 4,338,397.
In addition to control sequences, it may be desirable to add
regulatory sequences which allow for regulation of the expression
of the sequences relative to the growth of the host cell.
Regulatory sequences are known to those of skill in the art, and
examples include those which cause the expression of a gene to be
turned on or off in response to a chemical or physical stimulus,
including the presence of a regulatory compound. Other types of
regulatory elements may also be present in the vector. For example,
enhancer elements may be used herein to increase expression levels
of the constructs. Examples include the SV40 early gene enhancer
(Dijkema et al. (1985) EMBO J. 4:761), the enhancer/promoter
derived from the long terminal repeat (LTR) of the Rous Sarcoma
Virus (Gorman et al. (1982) Proc. Natl. Acad. Sci. USA 79:6777) and
elements derived from human CMV (Boshart et al. (1985) Cell
41:521), such as elements included in the CMV intron A sequence
(U.S. Pat. No. 5,688,688). The expression cassette may further
include an origin of replication for autonomous replication in a
suitable host cell, one or more selectable markers, one or more
restriction sites, a potential for high copy number and a strong
promoter.
An expression vector is constructed so that the particular coding
sequence is located in the vector with the appropriate regulatory
sequences, the positioning and orientation of the coding sequence
with respect to the control sequences being such that the coding
sequence is transcribed under the "control" of the control
sequences (i.e., RNA polymerase which binds to the DNA molecule at
the control sequences transcribes the coding sequence).
Modification of the sequences encoding the molecule of interest may
be desirable to achieve this end. For example, in some cases it may
be necessary to modify the sequence so that it can be attached to
the control sequences in the appropriate orientation; i.e., to
maintain the reading frame. The control sequences and other
regulatory sequences may be ligated to the coding sequence prior to
insertion into a vector. Alternatively, the coding sequence can be
cloned directly into an expression vector which already contains
the control sequences and an appropriate restriction site.
As explained above, it may also be desirable to produce mutants or
analogs of the antigen of interest. This is particularly true with
NS3/4a. Methods for doing so are described in, e.g., Dasmahapatra
et al., U.S. Pat. No. 5,843,752 and Zhang et al., U.S. Pat. No.
5,990,276. Mutants or analogs of this and other HCV proteins for
use in the subject assays may be prepared by the deletion of a
portion of the sequence encoding the polypeptide of interest, by
insertion of a sequence, and/or by substitution of one or more
nucleotides within the sequence. Techniques for modifying
nucleotide sequences, such as site-directed mutagenesis, and the
like, are well known to those skilled in the art. See, e.g.,
Sambrook et al., supra; Kunkel, T. A. (1985) Proc. Natl. Acad. Sci.
USA (1985) 82:448; Geisselsoder et al. (1987) BioTechniques 5:786;
Zoller and Smith (1983) Methods Enzymol. 100:468; Dalbie-McFarland
et al. (1982) Proc. Natl. Acad. Sci USA 79:6409.
The molecules can be expressed in a wide variety of systems,
including insect, mammalian, bacterial, viral and yeast expression
systems, all well known in the art.
For example, insect cell expression systems, such as baculovirus
systems, are known to those of skill in the art and described in,
e.g., Summers and Smith, Texas Agricultural Experiment Station
Bulletin No. 1555 (1987). Materials and methods for
baculovirus/insect cell expression systems are commercially
available in kit form from, inter alia, Invitrogen, San Diego
Calif. ("MaxBac" kit). Similarly, bacterial and mammalian cell
expression systems are well known in the art and described in,
e.g., Sambrook et al., supra. Yeast expression systems are also
known in the art and described in, e.g., Yeast Genetic Engineering
(Barr et al., eds., 1989) Butterworths, London.
A number of appropriate host cells for use with the above systems
are also known. For example, mammalian cell lines are known in the
art and include immortalized cell lines available from the American
Type Culture Collection (ATCC), such as, but not limited to,
Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney
(BHK) cells, monkey kidney cells (COS), human embryonic kidney
cells, human hepatocellular carcinoma cells (e.g., Hep G2),
Madin-Darby bovine kidney ("MDBK") cells, as well as others.
Similarly, bacterial hosts such as E. coli, Bacillus subtili, and
Streptococcus spp., will find use with the present expression
constructs. Yeast hosts useful in the present invention include
inter alia, Saccharomyces cerevisiae, Candida albicans, Candida
maltosa, Hansenula polymorpha, Kluyveromyces fragilis,
Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,
Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for
use with baculovirus expression vectors include, inter alia, Aedes
aegypti, Autographa californica, Bombyx mori, Drosophila
melanogaster, Spodoptera frugiperda, and Trichoplusia ni.
Nucleic acid molecules comprising nucleotide sequences of interest
can be stably integrated into a host cell genome or maintained on a
stable episomal element in a suitable host cell using various gene
delivery techniques well known in the art. See, e.g., U.S. Pat. No.
5,399,346.
Depending on the expression system and host selected, the molecules
are produced by growing host cells transformed by an expression
vector described above under conditions whereby the protein is
expressed. The expressed protein is then isolated from the host
cells and purified. If the expression system secretes the protein
into growth media, the product can be purified directly from the
media. If it is not secreted, it can be isolated from cell lysates.
The selection of the appropriate growth conditions and recovery
methods are within the skill of the art.
The production of various HCV antigens, including antigens used in
the multiple epitope fusion proteins described above, has been
described. See, e.g., Houghton et al., U.S. Pat. Nos. 5,350,671 and
5,683,864; Chien et al., J. Gastroent. Hepatol. (1993) 8:S33 39;
Chien et al., International Publication No. WO 93/00365; Chien, D.
Y., International Publication No. WO 94/01778; Chien et al., Proc.
Natl. Acad. Sci. USA (1992) 89:10011 10015; Chien, D. Y.,
International Publication No. WO 94/01778; and commonly owned,
allowed U.S. patent application Ser. Nos. 08/403,590 and
08/444,818, the disclosures of which are incorporated herein by
reference in their entireties.
Immunodiagnostic Assays
Once produced, the HCV antigens may be used in virtually any assay
format that employs a known antigen to detect antibodies. A common
feature of all of these assays is that the antigen is contacted
with the body component suspected of containing HCV antibodies
under conditions that permit the antigen to bind to any such
antibodies present in the component. Such conditions will typically
be physiologic temperature, pH and ionic strength using an excess
of antigen. The incubation of the antigen with the specimen is
followed by detection of immune complexes comprised of the
antigen.
Design of the immunoassays is subject to a great deal of variation,
and many formats are known in the art. Protocols may, for example,
use solid supports, or immunoprecipitation. Most assays involve the
use of labeled antibody or polypeptide; the labels may be, for
example, enzymatic, fluorescent, chemiluminescent, radioactive, or
dye molecules, as discussed in detail above. Assays which amplify
the signals from the immune complex are also known; examples of
which are assays which utilize biotin and avidin, and
enzyme-labeled and mediated immunoassays, such as ELISA assays.
The immunoassay may be, without limitation, a heterogenous or a
homogeneous format, and of a standard or competitive type. In a
heterogeneous format, the polypeptide is typically bound to a solid
matrix or support to facilitate separation of the sample from the
polypeptide after incubation. A solid support, for the purposes of
this invention, can be any material that is an insoluble matrix and
can have a rigid or semi-rigid surface. Exemplary solid supports
include, but are not limited to, substrates such as nitrocellulose
(e.g., in membrane or microtiter well form); polyvinylchloride
(e.g., sheets or microtiter wells); polystyrene latex (e.g., beads
or microtiter plates); polyvinylidine fluoride; diazotized paper;
nylon membranes; activated beads, magnetically responsive beads,
and the like. Particular supports include plates, pellets, disks,
capillaries, hollow fibers, needles, pins, solid fibers, cellulose
beads, pore-glass beads, silica gels, polystyrene beads optionally
cross-linked with divinylbenzene, grafted co-poly beads,
polyacrylamide beads, latex beads, dimethylacrylamide beads
optionally crosslinked with N-N'-bis-acryloylethylenediamine, and
glass particles coated with a hydrophobic polymer.
If desired, the molecules to be added to the solid support can
readily be functionalized to create styrene or acrylate moieties,
thus enabling the incorporation of the molecules into polystyrene,
polyacrylate or other polymers such as polyimide, polyacrylamide,
polyethylene, polyvinyl, polydiacetylene, polyphenylene-vinylene,
polypeptide, polysaccharide, polysulfone, polypyrrole,
polyimidazole, polythiophene, polyether, epoxies, silica glass,
silica gel, siloxane, polyphosphate, hydrogel, agarose, cellulose,
and the like.
In one context, a solid support is first reacted with the HCV
antigens (collectively called "the solid-phase components" herein),
under suitable binding conditions such that the molecules are
sufficiently immobilized to the support. Sometimes, immobilization
to the support can be enhanced by first coupling the antigen and/or
antibody to a protein with better solid phase-binding properties.
Suitable coupling proteins include, but are not limited to,
macromolecules such as serum albumins including bovine serum
albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules,
thyroglobulin, ovalbumin, and other proteins well known to those
skilled in the art. Other reagents that can be used to bind
molecules to the support include polysaccharides, polylactic acids,
polyglycolic acids, polymeric amino acids, amino acid copolymers,
and the like. Such molecules and methods of coupling these
molecules to antigens, are well known to those of ordinary skill in
the art. See, e.g., Brinkley, M. A. (1992) Bioconjugate Chem. 3:2
13; Hashida et al. (1984) J. Appl. Biochem. 6:56 63; and Anjaneyulu
and Staros (1987) International J. of Peptide and Protein Res.
30:117 124.
After reacting the solid support with the solid-phase components,
any nonimmobilized solid-phase components are removed from the
support by washing, and the support-bound components are then
contacted with a biological sample suspected of containing HCV
antibodies (collectively called "ligand molecules" herein) under
suitable binding conditions. If HCV antibodies are present in the
sample, they will form a complex with the HCV antigens. After
washing to remove any nonbound ligand molecules, detectably labeled
anti-xenogenic (e.g., anti-human) antibodies, which recognize an
epitope on anti-HCV antibodies, is added. These antibodies bind due
to complex formation.
In a homogeneous format, the test sample is incubated with the
combination of antigens in solution. For example, it may be under
conditions that will precipitate any antigen-antibody complexes
which are formed. Both standard and competitive formats for
homogeneous assays are also known in the art.
In a standard format, the amount of HCV antibodies forming the
antibody-antigen complex is directly monitored. This may be
accomplished by determining whether labeled anti-xenogenic (e.g.,
anti-human) antibodies which recognize an epitope on anti-HCV
antibodies will bind due to complex formation. In a competitive
format, the amount of HCV antibodies in the sample is deduced by
monitoring the competitive effect on the binding of a known amount
of labeled antibody (or other competing ligand) in the complex.
More particularly, complexes formed comprising anti-HCV antibody
(or, in the case of competitive assays, the amount of competing
antibody) are detected by any of a number of known techniques,
depending on the format. For example, unlabeled HCV antibodies in
the complex may be detected using a conjugate of antixenogeneic Ig
complexed with a label, (e.g., an enzyme label). In an
immunoprecipitation or agglutination assay format, the reaction
between the HCV antigens and the antibody forms a network that
precipitates from the solution or suspension and forms a visible
layer or film of precipitate. If no anti-HCV antibody is present in
the test specimen, no visible precipitate is formed.
The above-described assay reagents, including the immunoassay solid
support with bound antibodies and antigens, as well as antibodies
and antigens to be reacted with the captured sample, can be
provided in kits, with suitable instructions and other necessary
reagents, in order to conduct immunoassays as described above. The
kit will normally contain in separate containers the combination of
antigens (either already bound to a solid matrix or separate with
reagents for binding them to the matrix), control antibody
formulations (positive and/or negative), labeled antibody when the
assay format requires same and signal generating reagents (e.g.,
enzyme substrate) if the label does not generate a signal directly.
Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying
out the assay usually will be included in the kit. The kit can also
contain, depending on the particular immunoassay used, other
packaged reagents and materials (i.e. wash buffers and the like).
Standard immunoassays, such as those described above, can be
conducted using these kits.
III. Experimental
Below are examples of specific embodiments for carrying out the
present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g., amounts, temperatures, etc.), but some experimental
error and deviation should, of course, be allowed for.
EXAMPLE 1
Construction of MEFA 7 and MEFA 7.1
The following example illustrates the preparation of a polyprotein
cassette of multiple HCV epitopes. The polyprotein expressed from
the multiple epitope cassette is referred to herein as a Multiple
Epitope Fusion Antigen (MEFA). Preferably, where an epitope is
repeated, the extra copy or copies are tandemly arrayed in the same
orientation. It is understood that the region of a viral coding
sequence used as an epitope may be varied slightly and still retain
antigenic activity, and that the amino acid numbering designation
may vary from strain to strain. Thus, the repeated epitopes may
vary one from another in amino acid sequence due to strain sequence
variations and/or numbering designation. Preferably, the amino acid
sequences of repeated epitopes within a MEFA are at least 30%
homologous at the amino acid level, more preferably at least 40%
homologous at the amino acid level.
Unique restriction enzyme sites were introduced in order to connect
the epitopes in the prescribed order and enhance the usefulness of
the invention by facilitating modifications in design of a chimeric
antigen. The choice of restriction enzyme sites and cloning
procedures are readily determined by one of ordinary skill in the
art of recombinant DNA technology. Preferably, the epitope
junctions (amino acid sequences created between epitopes due to
cloning) do not generate non-specific epitopes. Non-specific
epitopes are, for example, non-HCV sequences which do not exist
adjacent to the HCV epitopes in nature. Non-specific epitopes may
bind antibodies in a test sample causing false positive assay
results. Preferably, the multiple epitope fusion protein is tested
for false positive results due to such sequences generated at the
epitope junctions. To avoid non-specific interactions with the MEFA
due to junction sequences, the DNA sequence encoding the junction
may, for example, be mutated such that non-specific interactions
with the mutant amino acid sequence are reduced, and cloning of the
epitope fragments is possible.
The HCV MEFA 7 and 7.1 expression cassettes were constructed by
cloning the coding nucleotide sequences containing major epitopes
in a tandem array as shown in FIG. 4. A major epitope was chosen
based on antibody reaction frequency and reaction intensity (titer)
to the epitope (Chein, D. Y. et al. (1994) Viral Hepatitis and
Liver Disease, pp. 320 324). The various DNA segments coding for
the HCV epitopes were constructed by PCR amplification or by
synthetic oligonucleotides. The amino acids in each segment are set
forth in Table 2 above and shown in FIGS. 5A 5F. The complete HCV-1
amino acid sequence (3011 amino acids) was determined by Choo, et
al. (1991) Proc. Natl. Acad. Sci. USA 88:2451 2455, herein
incorporated by reference in its entirety. Oligonucleotides capable
of binding to HCV are described in U.S. Pat. No. 5,350,671, herein
incorporated by reference in its entirety. The numbering of the
amino acids in epitopes of the invention follows the numbering
designation provided in Choo, et al., supra, in which amino acid
number 1 is the first methionine encoded by the coding sequence of
the core region, unless otherwise specified. For example, one
epitope segment from NS5 is represented by amino acids 2278 to 2313
of the HCV polyprotein. An epitope from the E1 region is
represented by amino acids 303 to 320, numbered relative to the
HCV-1 polyprotein.
MEFA 7 and 7.1 each contain epitopes from HCV-1, HCV-2 and HCV-3,
allowing for detection of multiple types of a virus in a single
assay. Methods of determining HCV serotype are found in WO
96/27153, herein incorporated by reference in its entirety. For
example, epitopes from the 5-1-1 region have been found to vary
between serotypes of HCV. A copy of each of the HCV type-specific
5-1-1 epitopes present in the MEFAs described herein allows binding
of any of the HCV types that may be present in the test biological
sample.
The MEFA 7 and 7.1 constructs were genetically engineered for
expression in Saccharomyces cerevisiae, utilizing the yeast
expression vector pBS24.1 which contains 2.mu. sequences for
autonomous replication in yeast and the yeast genes leu2-d and URA3
as selectable markers. The .beta.-lactamase gene and the ColE1
origin of replication, required for plasmid replication in
bacteria, were also present in this expression vector. The yeast
expression vector for MEFA 7, ps.MEFA7, was constructed first.
Subsequently, the plasmid was modified in the coding region for the
HCV core epitopes to create the plasmid ps.MEFA7.1, encoding the
MEFA 7.1 antigen.
In particular, as shown in FIGS. 7A through 7D, a yeast expression
plasmid for MEFA 7 was constructed as follows. First, a
BamHI/HindIII fragment of 1896 bp, encoding the ADH2/GAPDH hybrid
promoter, hSOD (amino acids 1 156), followed by an E1 epitope
(amino acids 303 320, HCV1 strain), was isolated from ps.MEFA6, the
expression plasmid encoding MEFA 6, described in International
Publication No. WO. 97/44469. Next, a HindIII/SphI synthetic DNA
fragment of 269 bp which contains the coding sequence for E2 HVR1a
consensus epitope (amino acids 390 410, HCV-1), E2 HVR1+2 consensus
epitope (amino acids 384 414, HCV1+2) and the 5' end of the
helicase domain (amino acids 1193 1229, HCV-1) was created. An
SphI/EclXI fragment of 1264 bp, encoding the remainder of the
helicase domain (amino acids 1230 1651, HCV-1), was gel-purified
from pTac5/HelI plasmid DNA. The HindIII/SphI synthetic DNA
fragment and the SphI/EclXI 1264 bp fragment were ligated into
vector pSP72new.HindIII/EclXI vector, to produce
pSP72new.HindIII/EclXI/e2.helicase. This vector was derived from
pSP72 an E. coli vector commercially available from Promega,
Madison, Wis. (see, GenBank/EMBL Accession Number X65332). In
particular, to facilitate the subcloning of several MEFA 7
epitopes, a new multiple cloning site (MCS) polylinker was
introduced, via synthetic oligos, between the SphI and BglII sites
of pSP72. This new plasmid, named pSP72new, was digested with
HindIII and EclXI (also known as EagI), which have unique sites in
the MCS. It was then dephosphorylated and gel-purified.
E. coli HB101 competent cells were transformed with the plasmid,
and plated on Luria agar plates containing 100 .mu.g/ml ampicillin.
Desired clones were identified using miniprep DNA analysis. After
sequence verification, the plasmid
pSP72new.HindIII/EclXI/e2.helicase subclone #4 was digested with
HindIII and EclXI(EagI) to generate a 1534 bp fragment. The
HindIII/EclX1 fragment was gel-purified and ligated with EclXI/SphI
oligonucleotides, encoding the last amino acids of the helicase
domain (amino acids 1651 1658, HCV-1), into a pGEM7 HindIII/SphI
vector. HB101 competent cells were transformed and plated on
Luria-ampicillin (100 .mu.g/ml). After identification of the
desired clones and sequence confirmation, pGEM7HindIII/SphI
subclone #9 was digested with HindIII and SphI to generate a 1560
bp fragment, which was gel purified (see, FIG. 7A).
To assemble the 3' end portion of MEFA 7, the following steps were
performed. The 5-1-1 epitopes (amino acids 1689 1735) from HCV-1,
HCV-3 and HCV-2 (in this order) were gel-isolated from ps.MEFA6,
the expression plasmid encoding MEFA 6, described in International
Publication No. WO 97/44469, as an SphI/AvaI fragment of 441 bp.
This fragment was ligated with synthetic AvaI/XbaI oligonucleotides
encoding the c100 epitope (amino acids 1901 1936) into a
pSP72new.SphI/XbaI vector. After HB101 transformation, clone
identification, and sequence verification, pSP72newSXi subclone #6
was digested with XbaI and NotI to prepare a pSP72newXbaI/NotI
vector. Additionally, an XbaI/NcoI fragment of 221 bp, which
encoded a double repeat of an NS5 epitope (amino acids 2278 2313,
HCV-1), was isolated from ps.MEFA6. The Xba/NcoI fragment was
ligated with NcoI/NotI oligonucleotides, encoding the first amino
acids of the HCV-1 core epitope, amino acids 9 17, in which the Lys
at position 9 was changed to Arg, and the Asn at position 11 was
changed to Thr, into the pSP72newXbaI/NotI vector prepared above.
HB101 transformants were analyzed and their plasmid DNA sequenced.
A subclone, termed pSP72newSX/XNi #3, was digested with NotI/SalI
to prepare a vector for subsequent subdloning (see, FIG. 7B).
To complete the assembly of the 3' end of MEFA 7, a double repeat
of the sequence encoding a core epitope with amino acids 9 53 from
HCV-1, plus two genotype-specific epitopes of the core region
(amino acids 64 88, HCV-1 and amino acids 67 84, HCV-2) were
subclone as follows into NotI-SalI digested pSP72newSX/XNi subclone
#3. First, a NotI/XmnI fragment of 92 bp encoding amino acids 18 51
of a core epitope was isolated from pd.Core191RT clone #20. Plasmid
pd.Core191RT was constructed by ligating into the pBS24.1
BamHI-SalI yeast expression vector, a 1365 bp BamHI-NcoI fragment
for the ADH2/GAPDH promoter, and a 615 bp NcoI-SalI fragment
encoding the first 191 amino acids of HCV-1 core with amino acid 9
mutated from Lys to Arg and amino acid 11 mutated from Asn to Thr.
The 615 bp NcoI-SalI fragment was derived from an E. coli
expression vector in which the core sequence for amino acids 1 191,
with the same two mutations described above, had been cloned.
The 92 bp NotI/XmnI fragment was ligated with a pSP72newNot/Kpn
vector and with XmnI/KpnI oligonucleotides which encode the 3' end
of the complete core epitope. After sequence verification of the
positive clones, pSP72newNKi subclone #4 was digested with NotI and
KpnI, and a 224 bp fragment was gel-isolated. This NotI/KpnI
fragment was ligated with 284 bp of oligonucleotides (KpnI-SalI
ends) encoding a complete repeat of the core epitope described
above into the pSP72newSX/XNi NotI/SalI vector described above.
After HB101 transformation, clone identification and sequence
verification, pSP72newSX/XN/NSi subclone #18 was digested with SphI
and SalI and a fragment of 1317 bp was gel-isolated (see, FIG.
7C).
Lastly, the following fragments, described above, were ligated into
the pBS24.1 BamHI/SalI yeast expression vector to create ps.MEFA7
(see, FIG. 7D):
the BamHI/HindIII fragment of 1896 bp (FIG. 7A)
the HindIII/SphI fragment of 1560 bp (FIG. 7A)
the SphI/SalI fragment of 1317 bp (FIG. 7C)
S. cerevisiae strain AD3 was transformed with ps.MEFA7 and single
transformants were checked for expression after depletion of
glucose in the medium. The recombinant protein was expressed at
high levels in yeast, as detected by Coomassie blue staining. In
particular, yeast cells were transformed with the MEFA expression
plasmid using a lithium acetate protocol. Ura transformants were
streaked for single colonies and patched onto Leu/8% glucose plates
to increase plasmid copy number. Leu starter cultures were grown
for 24 hours at 30.degree. C. and then diluted 1:20 in YEPD (yeast
extract bactopeptone 2% glucose) media. The cells were grown for 48
hours at 30.degree. C. and harvested. To test for expression of the
MEFA 7 recombinant antigen, an aliquot of the cells was lysed with
glass beads in lysis buffer (10 mM Tris-Cl pH 7.5, 1 mM EDTA, 10 mM
DTT). The lysate was centrifuged at high speed. The supernatant and
insoluble pellet were analyzed on SDS protein gels. MEFA 7 was
highly enriched in the insoluble pellet fraction.
The MEFA 7 antigen was purified as follows. S. cerevisiae cells
expressing MEFA 7 were harvested as described above. The cells were
suspended in lysis buffer (50 mM Tris, 0.15 M NaCl, 1 mM EDTA, 1 mM
PMSF, pH 8.0) and lysed in a Dyno-Mill (Wab Willy A. Bachofon,
Basel, Switzerland) or equivalent apparatus using glass beads. The
lysate was centrifuged at low speed conditions (3,000 to 5,000 rpm,
15 min) and the pellet containing the insoluble protein fraction
was washed with increasing concentrations of urea (1 M, 2 M, 3 M)
in lysis buffer. Protein was solubilized from the centrifugation
pellet with 0.1 N NaOH, 4 M urea in lysis buffer. Cell debris was
removed by low speed centrifugation at 3,000 to 5,000 rpm, 15 min.
The supernatant was adjusted to pH 8.0 with 6 N HCl to precipitate
proteins insoluble under these conditions.
The precipitate was removed by centrifugation and the supernatant
was adjusted to 2.3% SDS, 50 mM DTT, pH 8.0 and boiled for 3 min.
Proteins in the mixture were fractionated by gel filtration on a
Pharmacia Sephacryl S-400 in phosphate buffered saline containing
0.1% SDS, 1 mM EDTA and adjusted to pH 7.4. Column eluate fractions
containing MEFA 7 were collected, pooled, and concentrated on an
Amicon YM-30 membrane. Gel filtration was repeated on the pooled
fractions using the same column and conditions.
During the analysis of MEFA 7 in a trial assay, it was discovered
that a monoclonal antibody used as a detection conjugate reacted
with a specific sequence of the core epitope (amino acids 33 38).
Thus, ps.MEFA7.1 was designed to eliminate amino acids 33 38 from
the core epitope region.
A yeast expression vector for MEFA 7.1 was made as follows. First,
the double repeat of the core epitope at the 3' end of ps.MEFA7 was
modified. To do so, an NcoI/KpnI synthetic fragment of 206 bp,
encoding the first core epitope repeat (amino acids 9 32, 39 42 and
64 88 of HCV-1, and amino acids 67 82 of HCV-2), and a KpnI/SalI
synthetic fragment of 233 bp encoding amino acids 83 and 84 of
HCV-2, followed by the second core epitope repeat (amino acids 9
32, amino acids 39 42 and amino acids 64 88, HCV-1, amino acids 67
84, HCV-2) were subclone respectively into a pSP72new.NcoI/KpnI
vector and a pSP72new.KpnI/SalI vector. After HB101 transformation,
clone identification and sequence conformation, pSP72newNKi clone
#21 was digested with NcoI and KpnI to isolate the NcoI/KpnI
fragment of 206 bp and pSP72newKSi clone #32 was digested with KpnI
and SalI to isolate the KpnI/SalI fragment of 233 bp.
Plasmid ps.MEFA7.1 was assembled by ligating the following
fragments into the pBS24.1 BamHI/SalI yeast expression vector (see,
FIG. 8):
the BamHI/HindIII fragment of 1896 bp, described above for
ps.MEFA7;
the HindIII/SphI fragment of 1560 bp described above for
ps.MEFA7;
an SphI/NcoI fragment of 776 bp isolated from ps.MEFA7 encoding the
5-1-1 epitopes, c100 epitope and NS5 epitope;
the NcoI/KpnI fragment of 206 bp;
and KpnI/SalI fragment of 233 bp.
S. cerevisiae strain AD3 was transformed with ps.MEFA7.1 and single
transformants were checked for expression after depletion of
glucose in the medium, as described above. The recombinant protein
was expressed at high levels in yeast, as detected by Coomassie
blue staining.
The MEFA 7.1 antigen was purified as follows. S. cerevisiae cells
expressing MEFA 7.1 were harvested as described above. The cells
were suspended in lysis buffer (50 mM Tris, 0.15 M NaCl, 1 mM EDTA,
pH 8.0) and lysed in a Dyno-Mill (Wab Willy A. Bachofen, Basel,
Switzerland) or equivalent apparatus using glass beads. The lysate
was centrifuged at 10,000 rpm, 30 min in a JA-10 rotor, and the
pellet containing the insoluble protein fraction was washed with
increasing concentrations of Urea (1 M, 2 M, 3 M) in lysis buffer.
Protein was solubilized from the centrifugation pellet with 0.1 N
NaOH, 4 M Urea, 50 mM DTT in lysis buffer. Cell debris was removed
by centrifugation at 14,000 rpm, 20 min in a JA-14 rotor. The
supernatant was adjusted to pH 8.0 with 6 N HCl to precipitate
proteins insoluble under these conditions.
The precipitate was removed by centrifugation at 14,000 rpm, 20 min
in a JA-14 rotor. The supernatant was adjusted to 2.3% SDS and
heated to 70 75.degree. C. in boiling water then cooled to room
temperature. Proteins in the mixture were fractionated by gel
filtration on a Pharmacia Sephacryl S-400 HR in PBS containing 0.1%
SDS, 1 mM EDTA and adjusted to pH 7.4. Column eluate fractions
containing MEFA 7.1 were collected, pooled, and concentrated on an
Amicon YM-30 membrane. Pooled gel filtration fractions were
adjusted to 2.3% SDS, 50 mM DTT, and heated/cooled as above. This
pool was subjected to a second gel filtration step on a Pharmacia
Sephacryl S-300 HR column under the same conditions as the first
gel filtration step.
EXAMPLE 2
Recombinant Production of an NS3/4a Conformational Epitope
A conformational epitope of NS3/4a was obtained as follows. This
epitope has the sequence specified in FIGS. 3A through 3D and
differs from the native sequence at positions 403 (amino acid 1428
of the HCV-1 full-length sequence) and 404 (amino acid 1429 of the
HCV-1 full-length sequence). Specifically, the Thr normally
occurring at position 1428 of the native sequence has been mutated
to Pro and Ser which occurs at position 1429 of the native sequence
has been mutated to Ile.
In particular, the yeast expression vector used was pBS24.1,
described above. Plasmid pd.hcv1a.ns3ns4aPI, which encoded a
representative NS3/4a epitope used in the subject immunoassays, was
produced as follows. A two step procedure was used. First, the
following DNA pieces were ligated together: (a) synthetic
oligonucleotides which would provide a 5' HindIII cloning site,
followed by the sequence ACAAAACAAA (SEQ ID NO: 6), the initiator
ATG, and codons for HCV1a, beginning with amino acid 1027 and
continuing to a BglI site at amino acid 1046; (b) a 683 bp
BglI-ClaI restriction fragment (encoding amino acids 1046 1274)
from pAcHLTns3ns4aPI; and (c) a pSP72 vector (Promega, Madison,
Wis., GenBank/EMBL Accession Number X65332) which had been digested
with HindIII and ClaI, dephosphorylated, and gel-purified. Plasmid
pAcHLTns3ns4aPI was derived from pAcHLT, a baculovirus expression
vector commercially available from BD Pharmingen (San Diego,
Calif.). In particular, a pAcHLT EcoRI-PstI vector was prepared, as
well as the following fragments: EcoRI-AiwnI, 935 bp, corresponding
to amino acids 1027 1336 of the HCV-1 genome; AlwnI-SacII, 247 bp,
corresponding to amino acids 1336 1419 of the HCV-1 genome;
HinfI-BglI, 175 bp, corresponding to amino acids 1449 1509 of the
HCV-1 genome; BglI-PstI, 619 bp, corresponding to amino acids 1510
1711 of the HCV-1 genome, plus the transcription termination codon.
A SacII-HinfI synthetically generated fragment of 91 bp,
corresponding to amino acids 1420 1448 of the HCV-1 genome and
containing the PI mutations (Thr-1428 mutated to Pro, Ser-1429
mutated to Ile), was ligated with the 175 bp HinfI-BglI fragment
and the 619 bp BglI-PstI fragment described above and subclone into
a pGEM-5Zf(+) vector digested with SacII and PstI. pGEM-5Zf(+) is a
commercially available E. coli vector (Promega, Madison, Wis.,
GenBank/EMBL Accession Number X65308). After transformation of
competent HB101 cells, miniscreen analysis of individual clones and
sequence verification, an 885 bp SacII-PstI fragment from pGEM5.PI
clone2 was gel-purified. This fragment was ligated with the
EcoRI-AlwnI 935 bp fragment, the AlwnI-SacII 247 bp fragment and
the pAcHLT EcoRI-PstI vector, described above. The resultant
construct was named pAcHLTns3ns4aPI.
The ligation mixture above was transformed into HB101-competent
cells and plated on Luria agar plates containing 100 .mu.g/ml
ampicillin. Miniprep analyses of individual clones led to the
identification of putative positives, two of which were amplified.
The plasmid DNA for pSP72 1aHC, clones #1 and #2 were prepared with
a Qiagen Maxiprep kit and were sequenced.
Next, the following fragments were ligated together: (a) a 761 bp
HindIII-ClaI fragment from pSP721aHC #1 (pSP72.1aHC was generated
by ligating together the following: pSP72 which had been digested
with HindIII and ClaI, synthetic oligonucleotides which would
provide a 5' HindIII cloning site, followed by the sequence
ACAAAACAAA (SEC ID NO:6), the initiation codon ATG, and codons for
HCV1a, beginning with amino acid 1027 and continuing to a BglII
site at amino acid 1046, and a 683 bp BglII-ClaI restriction
fragment (encoding amino acids 1046 1274) from pAcHLTns3ns4aPI);
(b) a 1353 bp BamHI-HindIII fragment for the yeast hybrid promoter
ADH2/GAPDH; (c) a 1320 bp ClaI-SalI fragment (encoding HCV1a amino
acids 1046 1711 with Thr 1428 mutated to Pro and Ser 1429 mutated
to Ile) from pAcHLTns3ns4aPI; and (d) the pBS24.1 yeast expression
vector which had been digested with BamHI and SalI,
dephosphorylated and gel-purified. The ligation mixture was
transformed into competent HB101 and plated on Luria agar plates
containing 100 .mu.g/ml ampicillin. Miniprep analyses of individual
colonies led to the identification of clones with the expected 3446
bp BamHI-SalI insert which was comprised of the ADH2/GAPDH
promoter, the initiator codon ATG and HCV1a NS3/4a from amino acids
1027 1711 (shown as amino acids 1 686 of FIGS. 3A 3D), with Thr
1428 (amino acid position 403 of FIGS. 3A 3D) mutated to Pro and
Ser 1429 (amino acid position 404 of FIGS. 3A 3D) mutated to Ile.
The construct was named pd.HCV1a.ns3ns4aPI (see, FIG. 9).
S. cerevisiae strain AD3 was transformed with pd.HCV1a.ns3ns4aPI
and single transformants were checked for expression after
depletion of glucose in the medium. The recombinant protein was
expressed at high levels in yeast, as detected by Coomassie blue
staining and confirmed by immunoblot analysis using a polyclonal
antibody to the helicase domain of NS3.
EXAMPLE 3
Purification of NS3/4a Conformational Epitope
The NS3/4a conformational epitope was purified as follows. S.
cerevisiae cells from above, expressing the NS3/4a epitope were
harvested as described above. The cells were suspended in lysis
buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.1
.mu.M pepstatin, 1 .mu.M leupeptin) and lysed in a Dyno-Mill (Wab
Willy A. Bachofon, Basel, Switzerland) or equivalent apparatus
using glass beads, at a ratio of 1:1:1 cells:buffer:0.5 mm glass
beads. The lysate was centrifuged at 30100.times.g for 30 min at
4.degree. C. and the pellet containing the insoluble protein
fraction was added to wash buffer (6 ml/g start cell pellet weight)
and rocked at room temperature for 15 min. The wash buffer
consisted of 50 mM NaPO.sub.4 pH 8.0, 0.3 M NaCl, 5 mM
.beta.-mercaptoethanol, 10% glycerol, 0.05% octyl glucoside, 1 mM
EDTA, 1 mM PMSF, 0.1 .mu.M pepstatin, 1 .mu.M leupeptin. Cell
debris was removed by centrifugation at 30100.times.g for 30 min at
4.degree. C. The supernatant was discarded and the pellet
retained.
Protein was extracted from the pellet as follows. 6 ml/g extraction
buffer was added and rocked at room temperature for 15 min. The
extraction buffer consisted of 50 mM Tris pH 8.0, 1 M NaCl, 5 mM
.beta.-mercaptoethanol, 10% glycerol, 1 mM EDTA, 1 mM PMSF, 0.1
.mu.M pepstatin, 1 .mu.M leupeptin. This was centrifuged at
30100.times.g for 30 min at 4.degree. C. The supernatant was
retained and ammonium sulfate added to 17.5% using the following
formula: volume of supernatant (ml) multiplied by x % ammonium
sulfate/(1-x % ammonium sulfate)=ml of 4.1 M saturated ammonium
sulfate to add to the supernatant. The ammonium sulfate was added
dropwise while stirring on ice and the solution stirred on ice for
10 min. The solution was centrifuged at 17700.times.g for 30 min at
4.degree. C. and the pellet retained and stored at 2.degree. C. to
8.degree. C. for up to 48 hrs.
The pellet was resuspended and run on a Poly U column (Poly U
Sepharose 4B, Amersham Pharmacia) at 4.degree. C. as follows.
Pellet was resuspended in 6 ml Poly U equilibration buffer per gram
of pellet weight. The equilibration buffer consisted of 25 mM HEPES
pH 8.0, 200 mM NaCl, 5 mM DTT (added fresh), 10% glycerol, 1.2
octyl glucoside. The solution was rocked at 4.degree. C. for 15 min
and centrifuged at 31000.times.g for 30 min at 4.degree. C.
A Poly U column (1 ml resin per gram start pellet weight) was
prepared. Linear flow rate was 60 cm/hr and packing flow rate was
133% of 60 cm/hr. The column was equilibrated with equilibration
buffer and the supernatant of the resuspended ammonium sulfate
pellet was loaded onto the equilibrated column. The column was
washed to baseline with the equilibration buffer and protein eluted
with a step elution in the following Poly U elution buffer: 25 mM
HEPES pH 8.0, 1 M NaCl, 5 mM DTT (added fresh), 10% glycerol, 1.2
octyl glucoside. Column eluate was run on SDS-PAGE (Coomassie
stained) and aliquots frozen and stored at -80.degree. C. The
presence of the NS3/4a epitope was confirmed by Western blot, using
a polyclonal antibody directed against the NS3 protease domain and
a monoclonal antibody against the 5-1-1 epitope (HCV 4a).
Additionally, protease enzyme activity was monitored during
purification as follows. An NS4A peptide (KKGSVVIVGRIVLSGKPAIIPKK)
(SEQ ID NO:7), and the sample containing the NS3/4a conformational
epitope, were diluted in 90 .mu.l of reaction buffer (25 mM Tris,
pH 7.5, 0.15M NaCl, 0.5 mM EDTA, 10% glycerol, 0.05 n-Dodecyl
B-D-Maltoside, 5 mM DTT) and allowed to mix for 30 minutes at room
temperature. 90 .mu.l of the mixture were added to a microtiter
plate (Costar, Inc., Corning, N.Y.) and 10 .mu.l of HCV substrate
(AnaSpec, Inc., San Jose Calif.) was added. The plate was mixed and
read on a Fluostar plate reader. Results were expressed as relative
fluorescence units (RFU) per minute.
Using these methods, the product of the 1 M NaCl extraction
contained 3.7 RFU/min activity, the ammonium sulfate precipitate
had an activity of 7.5 RFU/min and the product of the Poly U
purification had an activity of 18.5 RFU/min.
EXAMPLE 4
Coating Solid Support with the HCV Antigens
The HCV NS3/4a conformational epitope and MEFA 7.1 antigen were
coated onto plates as follows. HCV coating buffer (50 mM NaPO4 pH
7.0, 2 mM EDTA and 0.1% Chloroacetamide) was filtered through a
0.22.mu. filter unit. The following reagents were then added
sequentially to the HCV coating buffer and stirred after each
addition: 2 .mu.g/ml BSA-Sulfhydryl Modified, from a 10 mg/ml
solution (Bayer Corp. Pentex, Kankakee, Ill.); 5 mM DTT from a 1 M
solution (Sigma, St. Louis, Mo.); 0.45 .mu.g/ml NS3/4a (protein
concentration of 0.3 mg/ml); 0.375 .mu.g/ml MEFA 7.1 (protein
concentration of 1 mg/ml). The final solution was stirred for 15
minutes at room temperature.
200 .mu.l of the above solution was added to each well of a Costar
high binding, flat bottom plate (Coming Inc., Coming, N.Y.) and the
plates were incubated overnight in a moisture chamber. The plates
were then washed with wash buffer (1.times.PBS, 0.1% TWEEN-20),
Tapped dry and 285 .mu.l Ortho Post-Coat Buffer (1.times.PBS, pH
7.4, 1% BSA, 3% sucrose) added. The plates were incubated for at
least 1 hour, tapped and dried overnight at 2 8.degree. C. The
plates were pouched with desiccants for future use.
EXAMPLE 5
Early Seroconversion Studies
The performance of the NS3/4a and MEFA 7.1 antigens in a
combination assay (HCV 4.0) was compared to other HCV assays to
test the seroconversion detection limits and to compare these
limits to those obtained in other commercially available assays.
Panels of commercially available human blood samples were used
which were HCV-infected. The PHV panels shown in the tables below
were purchased from Boston Biomedica, Inc., West Bridgewater, Mass.
(BBI). The 6212 panels were purchased from Bioclinical Partners,
Franklin, Mass. (BCP). The SC panels were purchased from North
American Biologics, Inc., BocoRatan, Fla. (NABI). The day on which
the blood samples was obtained is indicated in the tables.
The HCV 4.0 assay was conducted as follows. 200 .mu.l of specimen
diluent buffer (1 g/l casein, 100 mg/l recombinant human SOD, 1 g/l
chloracetamide, 10 g/l BSA, 500 mg/l yeast extract, 0.366 g/l EDTA,
1.162 g/l KPO.sub.4, 5 ml/i Tween-20, 29.22 g/l NaCl, 1.627 g/l
NaPO.sub.4, 1% SDS) was added to the coated plates. 20 .mu.l of
sample was then added. This was incubated at 37.degree. C. for one
hour. The plates were washed with wash buffer (1.times.PBS, pH 7.4,
0.1% Tween-20). 200 .mu.l conjugate solution (a mouse anti-human
IgG-HRP, such as mouse anti-human IgG-HRP diluted 1:22,000 in ORTHO
HCV 3.0 ELISA Test System with Enhanced SAVe bulk conjugate diluent
(Ortho-Clinical Diagnostics, Raritan, N.J.) was added and incubated
for 60 minutes at 37.degree. C. This was washed as above, and 200
.mu.l substrate solution (1 OPD tablet/10 ml) was added. The OPD
tablet contains o-phenylenediamine dihydrochloride and hydrogen
peroxide for horse radish peroxidase reaction color development.
This was incubated for 30 minutes at room temperature in the dark.
The reaction was stopped by addition of 50 .mu.l 4N H.sub.2SO.sub.4
and the plates were read at 492 nm, relative to absorbance at 690
nm as control.
The other assays used in the study were as follows:
The Abbott PRISM assay (Abbott Laboratories, Abbott Park, Ill.), is
commercially available and is an antibody-based detection assay.
The assay was performed using the manufacturer's instructions.
The ORTHO HCV Version 3.0 ELISA Test System (HCV 3.0) (Ortho
Clinical Diagnostics, Raritan, N.J.) is an antibody-based detection
assay. The assay was conducted using the manufacturer's
instructions.
The Pasteur MONOLISA anti-HCV Plus Version 2 assay (Sanofi
Diagnostics Pasteur, Marnes-la-Coquette, France) is an
antibody-based detection assay. The assay was performed using the
manufacturer's instructions.
The performance of the HCV 4.0 assay was compared to the HCV 3.0,
PRISM and Pasteur assays (see, Tables 3 and 4). HCV antibodies
present in the blood panels (anti-c33c or anti-c22) are set forth
in Table 4. In particular, 17 seroconversion panels from the three
commercial sources set forth above were assayed using the
techniques above. As can be seen, for the c33c panels, HCV 4.0
showed earlier detection (by 1 3 bleeds) than HCV 3.0 in 9 out of 9
c33c panels, and earlier detection than PRISM in 6 out of 9 panels
and equivalent detection as compared with PRISM in 3 of the 9
panels. For the c22 panels, HCV 4.0 showed earlier detection than
HCV 3.0 in 3 of 8 panels and equivalent detection in the other 5
panels. HCV 4.0 also showed earlier detection than PRISM in 2 of 8
panels and equivalent detection in 6 of 8 panels. The range of
improvement seen was 2 14 days over both the HCV 3.0 and PRISM
assays.
TABLE-US-00004 TABLE 3 ##STR00001## ##STR00002## ##STR00003##
##STR00004## ##STR00005## ##STR00006##
TABLE-US-00005 TABLE 4 Bleed days earlier detection Abbott early
c33c early c22 Panel HCV 3.0 Prism panels panels #1 c33c PHV 904 2
2 PHV 904 PHV 907 #2 c33c PHV 905 7 3 PHV 905 PHV 909 #3 c22 PHV
907 3 0 PHV 908 PHV 910 #4 c33c PHV 908 8 0 PHV 914 PHV 911 #5 c22
PHV 909 0 0 6212 PHV 912 #6 c22 PHV 910 0 0 6213 PHV 913 #7 c22 PHV
911 0 0 6214 SC-0010 #8 c22 PHV 912 0 0 6222 SC-0030 #9 c22 PHV 913
2 2 SC-0040 #10 c33c PHV 914 12 12 #11 c33c 6212 14 12 #12 c33c
6213 6 0 #13 c33c 6214 7 0 #14 c33c 6222 4 4 #15 c22 SC-0010 0 0
#16 c22 SC-0030 5 5 #17 c33c SC-0040 9 7 Range of improvement 2 14
days 2 12 days
EXAMPLE 6
HCV 4.0 Genotype Sensitivity
The genotype sensitivity of the HCV 4.0 assay was compared to the
HCV 3.0 and Pasteur assays, described above. In particular, samples
from 10 different HCV genotypes, specified in Table 5, were diluted
as indicated in the table (2-fold or 10-fold depending on initial
sample titering) and used in the three assays, using the procedures
described above. All three tests were run simultaneously. The data
is shown as signal or raw O.D. The data suggests that the HCV 4.0
prototype is more sensitive in detecting diluted genotype
samples.
TABLE-US-00006 TABLE 5 HCV 4.0 and Genotype Dilutional Sensitivity
HCV 4.0 HCV 3.0 Monolisa Ver. 2 prototype Ortho Pasteur dilution
genotype s s s 1:2500 1b 2.866 1.007 0.694 1:5000 2.074 0.393 0.218
1:10000 1.099 0.159 0.084 1:20000 0.403 0.045 0.028 1:2500 2b 1.430
0.295 0.658 1:5000 0.551 0.108 0.207 1:10000 0.225 0.032 0.061
1:20000 0.074 0.010 0.019 1:2500 2a/c 1.952 0.467 1.653 1:5000
0.917 0.136 0.782 1:10000 0.395 0.049 0.286 1:20000 0.108 0.011
0.105 1:2500 3a 2.580 0.514 0.941 1:5000 1.622 0.218 0.353 1:10000
0.873 0.067 0.164 1:20000 0.398 0.023 0.050 1:2500 3e 1.207 0.158
0.291 1:5000 0.461 0.039 0.114 1:10000 0.155 0.011 0.053 1:20000
0.054 0.003 0.024 4b/c 2.478 0.701 0.551 1.125 0.256 0.195 0.609
0.087 0.076 0.216 0.035 0.033 4a 2.831 0.632 0.462 1.752 0.193
0.181 0.717 0.069 0.076 0.248 0.015 0.025 4c 1.751 0.457 1.147
0.856 0.169 0.474 0.384 0.055 0.178 0.141 0.018 0.058 5a 2.682
1.496 2.271 2.744 0.827 0.988 1.587 0.316 0.395 0.726 0.097 0.120
1:10 6 3.516 3.247 ND 1:100 3.602 3.594 ND 1:1000 3.224 2.863 ND
1:10000 1.192 0.380 ND
EXAMPLE 7
Competition Studies
The following competition study was conducted in order to assess
whether the NS3/4a conformational epitope detected different
antibodies than other HCV antigens. In particular, the NS3/4a
antigen was compared with the c200 antigen as follows.
0.5 .mu.g and 1.0 .mu.g of NS3/4a, produced as described above, or
c200 (Hepatology (1992) 15:19 25, available in the ORTHO HCV
Version 3.0 ELISA Test System, Ortho-Clinical Diagnostics, Raritan,
N.J.), were mixed with 20 .mu.l of sample PHV914-5 (an early
seroconversion bleed obtained from blood of an infected individual)
in a total volume of 220 .mu.l (1.times.PBS). The mixture was
incubated for 1 hour in microwells at 37.degree. C. The mixture was
then transferred to NS3/4a-coated plates and incubated for 1 hour
at 37.degree. C. Plates were washed and assayed as follows.
1 .mu.g of c200 antigen was added to 10 .mu.l of sample PHV914-5 in
a total volume of about 220 .mu.l. The mixture was incubated for 1
hour in a micro well at 37.degree. C. and 200 .mu.l transferred to
an NS3/4a-coated plate (100 ng/assay) and incubated for 1 hour at
37.degree. C. Plates were washed five times with 1.times.PBS, 0.1%
Tween-20. 200 .mu.l of conjugate solution (described above) were
added, and the plates incubated and assayed as described in Example
4 for the HCV 4.0 assay. Controls which consisted of PHV914-5 and
1.times.PBS (without antigen) were also treated as above.
Results are shown in Table 6. Percent inhibition results shown in
column 4 are calculated as column 3 minus (column 2 divided by
column 3 times 100). As can be seen, the data show that NS34a is
neutralized by early seroconversion antibodies and c200 is not. A
strong signal was achieved when antibodies in PHV914-5 c33c early
seroconversion panel member reacted with the NS34a coated on the
plate. The c200 antigen was not neutralized by these antibodies.
This is shown in the top panel of Table 6. When NS34a was mixed
with the PHV914-5 sample, it was neutralized and therefore no
antibodies were present in the sample to react with NS34a that was
coated on the microplate. The data indicate that NS34a may be
detecting a different class of antibodies than is detected by
c200.
Competition Studies to Show NS34a Antigen Detects Different
Antibodies in Early c33c Seroconversion Panel Compared to c200
Antigen
TABLE-US-00007 TABLE 6 3 *Control 2 1xPBS 4 1 s s % Inhibition c200
+ PHV914-5 1 ug 1.450 1.645 12 1 ug 1.545 1.687 8 0.5 ug 1.557
1.913 19 0.5 ug 1.719 1.804 5 NS3/4a + PHV914-5 1 ug 0.054 1.599 97
1 ug 0.037 1.677 98 0.5 ug 0.066 1.672 96 0.5 ug NA 1.524 NA
EXAMPLE 8
Stability Studies of NS3/4a Conformational Epitope
To assess the role of stability of the NS3/4a epitope to assay
performance, the following study was done to determine NS3/4a
immunoreactivity versus time at room temperature. Small aliquots of
stock NS3/4a were allowed to sit at room temperature and then
frozen at intervals as shown in Table 7. All vials were coated
simultaneously and tested against two early NS3 seroconversion
panels. Assays were conducted as described above in Example 5 for
HCV 4.0.
As can be seen in Table 7, the NS3/4a stock is not stable and
immunoreactivity decreases with time. In addition, maintaining
NS3/4a conformation is necessary for immunoreactivity.
Further stability studies were conducted as follows. Two
conformational monoclonal antibodies made against NS3/4a using
standard procedures were substituted for anti-HCV early
seroconversion panels. Stock NS3/4a vials were stored at room
temperature at time intervals 3, 6 and 24 hours. The NS3/4a from
the frozen vials was coated at 90 ng/ml and assayed using the
procedure described above. Results suggested that the two
monoclonals were indeed conformational and their reactivity was
sensitive to the handling of stock NS3/4a antigen at room
temperature. The reactivity of a positive control monoclonal
antibody did not change.
TABLE-US-00008 TABLE 7 ##STR00007## ##STR00008##
EXAMPLE 9
Immunoreactivity of NS3/4a Conformational Epitope Verus Denatured
NS3/4a
The immunoreactivity of the NS3/4a conformational epitope, produced
as described above, was compared to NS3/4a which had been denatured
by adding SDS to the NS3/4a conformational epitope preparation to a
final concentration of 2%. The denatured NS3/4a and conformational
NS3/4a were coated onto microtiter plates as described above. The
c200 antigen (Hepatology (1992) 15:19 25, available in the ORTHO
HCV Version 3.0 ELISA Test System, Ortho-Clinical Diagnostics,
Raritan, N.J.) was also coated onto microtiter plates. The c200
antigen was used as a comparison it is presumed to be
non-conformational due to the presence of reducing agent (DTT) and
detergent (SDS) in its formulation.
The immunoreactivity was tested against two early HCV
seroconversion panels, PHV 904 and PHV 914 (commercially available
human blood samples from Boston Biomedica, Inc., West Bridgewater,
Mass.), using the ELISA assay procedure described above. The
results are shown in Table 8. The data suggests that the denatured
or linearized form of NS3/4a (as well as c200) does not detect
early seroconversion panels as early as the NS3/4a conformational
epitope.
TABLE-US-00009 TABLE 8 ##STR00009## ##STR00010## ##STR00011##
Immunoreactivity of the conformational epitope was also tested
using monoclonal antibodies to NS3/4a, made using standard
procedures. These monoclonal antibodies were then tested in the
ELISA format described above against NS3/4a and denatured NS3/4a
and c200 antigen. The data show that anti-NS3/4a monoclonals react
to the NS3/4a and denatured NS3/4a in a similar manner to the
seroconversion panels shown in Table 9. This result also provides
further evidence that the NS3/4a is conformational in nature as
monoclonal antibodies can be made which are similar in reactivity
to the early c33c seroconversion panels.
TABLE-US-00010 TABLE 9 Plate NS3/4a dNS3/4a c200 Monoclonal OD OD
OD 4B9/E3 1:100 1.820 0.616 0.369 1:1000 1.397 0.380 0.246 1:10000
0.864 0.173 0.070 1:20000 0.607 0.116 0.085 5B7/D7 1:100 2.885
0.898 0.436 1:1000 2.866 0.541 0.267 1:10000 1.672 0.215 0.086
1:20000 1.053 0.124 0.059 1A8/H2 1:100 1.020 0.169 0.080 1:1000
0.921 0.101 0.043 1:10000 0.653 0.037 0.013 1:20000 0.337 0.027
0.011
Accordingly, novel HCV detection assays have been disclosed. From
the foregoing, it will be appreciated that, although specific
embodiments of the invention have been described herein for
purposes of illustration, various modifications may be made without
deviating from the spirit and scope thereof.
SEQUENCE LISTINGS
1
7 1 2058 DNA Artificial Sequence Description of Artificial Sequence
representative NS3/4a conformational antigen 1 atg gcg ccc atc acg
gcg tac gcc cag cag aca agg ggc ctc cta ggg 48 Met Ala Pro Ile Thr
Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly 1 5 10 15 tgc ata atc
acc agc cta act ggc cgg gac aaa aac caa gtg gag ggt 96 Cys Ile Ile
Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu Gly 20 25 30 gag
gtc cag att gtg tca act gct gcc caa acc ttc ctg gca acg tgc 144 Glu
Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu Ala Thr Cys 35 40
45 atc aat ggg gtg tgc tgg act gtc tac cac ggg gcc gga acg agg acc
192 Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly Ala Gly Thr Arg Thr
50 55 60 atc gcg tca ccc aag ggt cct gtc atc cag atg tat acc aat
gta gac 240 Ile Ala Ser Pro Lys Gly Pro Val Ile Gln Met Tyr Thr Asn
Val Asp 65 70 75 80 caa gac ctt gtg ggc tgg ccc gct ccg caa ggt agc
cga tca ttg aca 288 Gln Asp Leu Val Gly Trp Pro Ala Pro Gln Gly Ser
Arg Ser Leu Thr 85 90 95 ccc tgc act tgc ggc tcc tcg gac ctt tac
ctg gtc acg agg cac gcc 336 Pro Cys Thr Cys Gly Ser Ser Asp Leu Tyr
Leu Val Thr Arg His Ala 100 105 110 gat gtc att ccc gtg cgc cgg cgg
ggt gat agc agg ggc agc ctg ctg 384 Asp Val Ile Pro Val Arg Arg Arg
Gly Asp Ser Arg Gly Ser Leu Leu 115 120 125 tcg ccc cgg ccc att tcc
tac ttg aaa ggc tcc tcg ggg ggt ccg ctg 432 Ser Pro Arg Pro Ile Ser
Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu 130 135 140 ttg tgc ccc gcg
ggg cac gcc gtg ggc ata ttt agg gcc gcg gtg tgc 480 Leu Cys Pro Ala
Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys 145 150 155 160 acc
cgt gga gtg gct aag gcg gtg gac ttt atc cct gtg gag aac cta 528 Thr
Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val Glu Asn Leu 165 170
175 gag aca acc atg agg tcc ccg gtg ttc acg gat aac tcc tct cca cca
576 Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro
180 185 190 gta gtg ccc cag agc ttc cag gtg gct cac ctc cat gct ccc
aca ggc 624 Val Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro
Thr Gly 195 200 205 agc ggc aaa agc acc aag gtc ccg gct gca tat gca
gct cag ggc tat 672 Ser Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala
Ala Gln Gly Tyr 210 215 220 aag gtg cta gta ctc aac ccc tct gtt gct
gca aca ctg ggc ttt ggt 720 Lys Val Leu Val Leu Asn Pro Ser Val Ala
Ala Thr Leu Gly Phe Gly 225 230 235 240 gct tac atg tcc aag gct cat
ggg atc gat cct aac atc agg acc ggg 768 Ala Tyr Met Ser Lys Ala His
Gly Ile Asp Pro Asn Ile Arg Thr Gly 245 250 255 gtg aga aca att acc
act ggc agc ccc atc acg tac tcc acc tac ggc 816 Val Arg Thr Ile Thr
Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly 260 265 270 aag ttc ctt
gcc gac ggc ggg tgc tcg ggg ggc gct tat gac ata ata 864 Lys Phe Leu
Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile Ile 275 280 285 att
tgt gac gag tgc cac tcc acg gat gcc aca tcc atc ttg ggc att 912 Ile
Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile 290 295
300 ggc act gtc ctt gac caa gca gag act gcg ggg gcg aga ctg gtt gtg
960 Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val
305 310 315 320 ctc gcc acc gcc acc cct ccg ggc tcc gtc act gtg ccc
cat ccc aac 1008 Leu Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val
Pro His Pro Asn 325 330 335 atc gag gag gtt gct ctg tcc acc acc gga
gag atc cct ttt tac ggc 1056 Ile Glu Glu Val Ala Leu Ser Thr Thr
Gly Glu Ile Pro Phe Tyr Gly 340 345 350 aag gct atc ccc ctc gaa gta
atc aag ggg ggg aga cat ctc atc ttc 1104 Lys Ala Ile Pro Leu Glu
Val Ile Lys Gly Gly Arg His Leu Ile Phe 355 360 365 tgt cat tca aag
aag aag tgc gac gaa ctc gcc gca aag ctg gtc gca 1152 Cys His Ser
Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala 370 375 380 ttg
ggc atc aat gcc gtg gcc tac tac cgc ggt ctt gac gtg tcc gtc 1200
Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val 385
390 395 400 atc ccg ccc atc ggc gat gtt gtc gtc gtg gca acc gat gcc
ctc atg 1248 Ile Pro Pro Ile Gly Asp Val Val Val Val Ala Thr Asp
Ala Leu Met 405 410 415 acc ggc tat acg ggc gac ttc gac tcg gtg ata
gac tgc aat acg tgt 1296 Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val
Ile Asp Cys Asn Thr Cys 420 425 430 gtc acc cag aca gtc gat ttc agc
ctt gac cct acc ttc acc att gag 1344 Val Thr Gln Thr Val Asp Phe
Ser Leu Asp Pro Thr Phe Thr Ile Glu 435 440 445 aca atc acg ctc ccc
caa gat gct gtc tcc cgc act caa cgt cgg ggc 1392 Thr Ile Thr Leu
Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly 450 455 460 agg act
ggc agg ggg aag cca ggc atc tac aga ttt gtg gca ccg ggg 1440 Arg
Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly 465 470
475 480 gag cgc ccc tcc ggc atg ttc gac tcg tcc gtc ctc tgt gag tgc
tat 1488 Glu Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu
Cys Tyr 485 490 495 gac gca ggc tgt gct tgg tat gag ctc acg ccc gcc
gag act aca gtt 1536 Asp Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro
Ala Glu Thr Thr Val 500 505 510 agg cta cga gcg tac atg aac acc ccg
ggg ctt ccc gtg tgc cag gac 1584 Arg Leu Arg Ala Tyr Met Asn Thr
Pro Gly Leu Pro Val Cys Gln Asp 515 520 525 cat ctt gaa ttt tgg gag
ggc gtc ttt aca ggc ctc act cat ata gat 1632 His Leu Glu Phe Trp
Glu Gly Val Phe Thr Gly Leu Thr His Ile Asp 530 535 540 gcc cac ttt
cta tcc cag aca aag cag agt ggg gag aac ctt cct tac 1680 Ala His
Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr 545 550 555
560 ctg gta gcg tac caa gcc acc gtg tgc gct agg gct caa gcc cct ccc
1728 Leu Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro
Pro 565 570 575 cca tcg tgg gac cag atg tgg aag tgt ttg att cgc ctc
aag ccc acc 1776 Pro Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg
Leu Lys Pro Thr 580 585 590 ctc cat ggg cca aca ccc ctg cta tac aga
ctg ggc gct gtt cag aat 1824 Leu His Gly Pro Thr Pro Leu Leu Tyr
Arg Leu Gly Ala Val Gln Asn 595 600 605 gaa atc acc ctg acg cac cca
gtc acc aaa tac atc atg aca tgc atg 1872 Glu Ile Thr Leu Thr His
Pro Val Thr Lys Tyr Ile Met Thr Cys Met 610 615 620 tcg gcc gac ctg
gag gtc gtc acg agc acc tgg gtg ctc gtt ggc ggc 1920 Ser Ala Asp
Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly 625 630 635 640
gtc ctg gct gct ttg gcc gcg tat tgc ctg tca aca ggc tgc gtg gtc
1968 Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val
Val 645 650 655 ata gtg ggc agg gtc gtc ttg tcc ggg aag ccg gca atc
ata cct gac 2016 Ile Val Gly Arg Val Val Leu Ser Gly Lys Pro Ala
Ile Ile Pro Asp 660 665 670 agg gaa gtc ctc tac cga gag ttc gat gag
atg gaa gag tgc 2058 Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met
Glu Glu Cys 675 680 685 2 686 PRT Artificial Sequence Description
of Artificial Sequence representative NS3/4a conformational antigen
2 Met Ala Pro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly 1
5 10 15 Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu
Gly 20 25 30 Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu
Ala Thr Cys 35 40 45 Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly
Ala Gly Thr Arg Thr 50 55 60 Ile Ala Ser Pro Lys Gly Pro Val Ile
Gln Met Tyr Thr Asn Val Asp 65 70 75 80 Gln Asp Leu Val Gly Trp Pro
Ala Pro Gln Gly Ser Arg Ser Leu Thr 85 90 95 Pro Cys Thr Cys Gly
Ser Ser Asp Leu Tyr Leu Val Thr Arg His Ala 100 105 110 Asp Val Ile
Pro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu Leu 115 120 125 Ser
Pro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro Leu 130 135
140 Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala Val Cys
145 150 155 160 Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val
Glu Asn Leu 165 170 175 Glu Thr Thr Met Arg Ser Pro Val Phe Thr Asp
Asn Ser Ser Pro Pro 180 185 190 Val Val Pro Gln Ser Phe Gln Val Ala
His Leu His Ala Pro Thr Gly 195 200 205 Ser Gly Lys Ser Thr Lys Val
Pro Ala Ala Tyr Ala Ala Gln Gly Tyr 210 215 220 Lys Val Leu Val Leu
Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly 225 230 235 240 Ala Tyr
Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly 245 250 255
Val Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr Gly 260
265 270 Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile
Ile 275 280 285 Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser Ile
Leu Gly Ile 290 295 300 Gly Thr Val Leu Asp Gln Ala Glu Thr Ala Gly
Ala Arg Leu Val Val 305 310 315 320 Leu Ala Thr Ala Thr Pro Pro Gly
Ser Val Thr Val Pro His Pro Asn 325 330 335 Ile Glu Glu Val Ala Leu
Ser Thr Thr Gly Glu Ile Pro Phe Tyr Gly 340 345 350 Lys Ala Ile Pro
Leu Glu Val Ile Lys Gly Gly Arg His Leu Ile Phe 355 360 365 Cys His
Ser Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala 370 375 380
Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val 385
390 395 400 Ile Pro Pro Ile Gly Asp Val Val Val Val Ala Thr Asp Ala
Leu Met 405 410 415 Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp
Cys Asn Thr Cys 420 425 430 Val Thr Gln Thr Val Asp Phe Ser Leu Asp
Pro Thr Phe Thr Ile Glu 435 440 445 Thr Ile Thr Leu Pro Gln Asp Ala
Val Ser Arg Thr Gln Arg Arg Gly 450 455 460 Arg Thr Gly Arg Gly Lys
Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly 465 470 475 480 Glu Arg Pro
Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr 485 490 495 Asp
Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr Val 500 505
510 Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln Asp
515 520 525 His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr His
Ile Asp 530 535 540 Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly Glu
Asn Leu Pro Tyr 545 550 555 560 Leu Val Ala Tyr Gln Ala Thr Val Cys
Ala Arg Ala Gln Ala Pro Pro 565 570 575 Pro Ser Trp Asp Gln Met Trp
Lys Cys Leu Ile Arg Leu Lys Pro Thr 580 585 590 Leu His Gly Pro Thr
Pro Leu Leu Tyr Arg Leu Gly Ala Val Gln Asn 595 600 605 Glu Ile Thr
Leu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys Met 610 615 620 Ser
Ala Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly Gly 625 630
635 640 Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val
Val 645 650 655 Ile Val Gly Arg Val Val Leu Ser Gly Lys Pro Ala Ile
Ile Pro Asp 660 665 670 Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met
Glu Glu Cys 675 680 685 3 3297 DNA Artificial Sequence Description
of Artificial Sequence MEFA 7.1 3 atg gct aca aag gct gtt tgt gtt
ttg aag ggt gac ggc cca gtt caa 48 Met Ala Thr Lys Ala Val Cys Val
Leu Lys Gly Asp Gly Pro Val Gln 1 5 10 15 ggt att att aac ttc gag
cag aag gaa agt aat gga cca gtg aag gtg 96 Gly Ile Ile Asn Phe Glu
Gln Lys Glu Ser Asn Gly Pro Val Lys Val 20 25 30 tgg gga agc att
aaa gga ctg act gaa ggc ctg cat gga ttc cat gtt 144 Trp Gly Ser Ile
Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val 35 40 45 cat gag
ttt gga gat aat aca gca ggc tgt acc agt gca ggt cct cac 192 His Glu
Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His 50 55 60
ttt aat cct cta tcc aga aaa cac ggt ggg cca aag gat gaa gag agg 240
Phe Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg 65
70 75 80 cat gtt gga gac ttg ggc aat gtg act gct gac aaa gat ggt
gtg gcc 288 His Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly
Val Ala 85 90 95 gat gtg tct att gaa gat tct gtg atc tca ctc tca
gga gac cat tgc 336 Asp Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser
Gly Asp His Cys 100 105 110 atc att ggc cgc aca ctg gtg gtc cat gaa
aaa gca gat gac ttg ggc 384 Ile Ile Gly Arg Thr Leu Val Val His Glu
Lys Ala Asp Asp Leu Gly 115 120 125 aaa ggt gga aat gaa gaa agt aca
aag aca gga aac gct gga agt cgt 432 Lys Gly Gly Asn Glu Glu Ser Thr
Lys Thr Gly Asn Ala Gly Ser Arg 130 135 140 ttg gct tgt ggt gta att
ggg atc gcc cag aat ttg aat tct ggt tgc 480 Leu Ala Cys Gly Val Ile
Gly Ile Ala Gln Asn Leu Asn Ser Gly Cys 145 150 155 160 aat tgc tct
atc tat ccc ggc cat ata acg ggt cac cgc atg gca tgg 528 Asn Cys Ser
Ile Tyr Pro Gly His Ile Thr Gly His Arg Met Ala Trp 165 170 175 aag
ctt ggt tcc gcc gcc aga act acc tcg ggc ttt gtc tcc ttg ttc 576 Lys
Leu Gly Ser Ala Ala Arg Thr Thr Ser Gly Phe Val Ser Leu Phe 180 185
190 gcc cca ggt gcc aaa caa aac gaa act cac gtc acg gga ggc gca gcc
624 Ala Pro Gly Ala Lys Gln Asn Glu Thr His Val Thr Gly Gly Ala Ala
195 200 205 gcc cga act acg tct ggg ttg acc tct ttg ttc tcc cca ggt
gcc agc 672 Ala Arg Thr Thr Ser Gly Leu Thr Ser Leu Phe Ser Pro Gly
Ala Ser 210 215 220 caa aac att caa ttg att gtc gac ttt atc cct gtg
gag aac cta gag 720 Gln Asn Ile Gln Leu Ile Val Asp Phe Ile Pro Val
Glu Asn Leu Glu 225 230 235 240 aca acc atg cga tct ccg gtg ttc acg
gat aac tcc tct cca cca gta 768 Thr Thr Met Arg Ser Pro Val Phe Thr
Asp Asn Ser Ser Pro Pro Val 245 250 255 gtg ccc cag agc ttc cag gtg
gct cac ctc cat gct ccc aca ggc agc 816 Val Pro Gln Ser Phe Gln Val
Ala His Leu His Ala Pro Thr Gly Ser 260 265 270 ggc aaa agc acc aag
gtc ccg gct gca tat gca gct cag ggc tat aag 864 Gly Lys Ser Thr Lys
Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr Lys 275 280 285 gtg cta gta
ctc aac ccc tct gtt gct gca aca ctg ggc ttt ggt gct 912 Val Leu Val
Leu Asn Pro Ser Val Ala Ala Thr Leu Gly Phe Gly Ala 290 295 300 tac
atg tcc aag gct cat ggg atc gat cct aac atc agg acc ggg gtg 960 Tyr
Met Ser Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr Gly Val 305 310
315 320 aga aca att acc act ggc agc ccc atc acg tac tcc acc tac ggc
aag 1008 Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr
Gly Lys 325 330 335 ttc ctt gcc gac ggc ggg tgc tcg ggg ggc gct tat
gac ata ata att 1056 Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala
Tyr Asp Ile Ile Ile 340 345 350 tgt gac gag tgc cac tcc acg gat gcc
aca tcc atc ttg ggc att ggc 1104 Cys Asp Glu Cys His Ser Thr Asp
Ala Thr Ser Ile Leu Gly Ile Gly 355 360 365 act gtc ctt gac caa gca
gag act gcg ggg gcg aga ctg gtt gtg ctc 1152 Thr Val Leu Asp Gln
Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu 370
375 380 gcc acc gcc acc cct ccg ggc tcc gtc act gtg ccc cat ccc aac
atc 1200 Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro
Asn Ile 385 390 395 400 gag gag gtt gct ctg tcc acc acc gga gag atc
cct ttt tac ggc aag 1248 Glu Glu Val Ala Leu Ser Thr Thr Gly Glu
Ile Pro Phe Tyr Gly Lys 405 410 415 gct atc ccc ctc gaa gta atc aag
ggg ggg aga cat ctc atc ttc tgt 1296 Ala Ile Pro Leu Glu Val Ile
Lys Gly Gly Arg His Leu Ile Phe Cys 420 425 430 cat tca aag aag aag
tgc gac gaa ctc gcc gca aag ctg gtc gca ttg 1344 His Ser Lys Lys
Lys Cys Asp Glu Leu Ala Ala Lys Leu Val Ala Leu 435 440 445 ggc atc
aat gcc gtg gcc tac tac cgc ggt ctt gac gtg tcc gtc atc 1392 Gly
Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser Val Ile 450 455
460 ccg acc agc ggc gat gtt gtc gtc gtg gca acc gat gcc ctc atg acc
1440 Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu Met
Thr 465 470 475 480 ggc tat acc ggc gac ttc gac tcg gtg ata gac tgc
aat acg tgt gtc 1488 Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile Asp
Cys Asn Thr Cys Val 485 490 495 acc cag aca gtc gat ttc agc ctt gac
cct acc ttc acc att gag aca 1536 Thr Gln Thr Val Asp Phe Ser Leu
Asp Pro Thr Phe Thr Ile Glu Thr 500 505 510 atc acg ctc ccc caa gat
gct gtc tcc cgc act caa cgt cgg ggc agg 1584 Ile Thr Leu Pro Gln
Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg 515 520 525 act ggc agg
ggg aag cca ggc atc tac aga ttt gtg gca ccg ggg gag 1632 Thr Gly
Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro Gly Glu 530 535 540
cgc ccc tcc ggc atg ttc gac tcg tcc gtc ctc tgt gag tgc tat gac
1680 Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys Glu Cys Tyr
Asp 545 550 555 560 gca ggc tgt gct tgg tat gag ctc acg ccc gcc gag
act aca gtt agg 1728 Ala Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala
Glu Thr Thr Val Arg 565 570 575 cta cga gcg tac atg aac acc ccg ggg
ctt ccc gtg tgc cag gac cat 1776 Leu Arg Ala Tyr Met Asn Thr Pro
Gly Leu Pro Val Cys Gln Asp His 580 585 590 ctt gaa ttt tgg gag ggc
gtc ttt aca ggc ctc act cat ata gat gcc 1824 Leu Glu Phe Trp Glu
Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala 595 600 605 cac ttt cta
tcc cag aca aag cag agt ggg gag aac ctt cct tac ctg 1872 His Phe
Leu Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu 610 615 620
gta gcg tac caa gcc acc gtg tgc gct agg gct caa gcc cct ccc cca
1920 Val Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro
Pro 625 630 635 640 tcg tgg gac cag atg tgg aag tgt ttg att cgc ctc
aag ccc acc ctc 1968 Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg
Leu Lys Pro Thr Leu 645 650 655 cat ggg cca aca ccc ctg cta tac aga
ctg ggc gct gtt cag aat gaa 2016 His Gly Pro Thr Pro Leu Leu Tyr
Arg Leu Gly Ala Val Gln Asn Glu 660 665 670 atc acc ctg acg cac cca
gtc acc aaa tac atc atg aca tgc atg tcg 2064 Ile Thr Leu Thr His
Pro Val Thr Lys Tyr Ile Met Thr Cys Met Ser 675 680 685 gcc gac ctg
gag gtc gtc acg agc gca tgc tcc ggg aag ccg gca atc 2112 Ala Asp
Leu Glu Val Val Thr Ser Ala Cys Ser Gly Lys Pro Ala Ile 690 695 700
ata cct gac agg gaa gtc ctc tac cga gag ttc gat gag atg gaa gag
2160 Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu Met Glu
Glu 705 710 715 720 tgc tct cag cac tta ccg tac atc gag caa ggg atg
atg ctc gcc gag 2208 Cys Ser Gln His Leu Pro Tyr Ile Glu Gln Gly
Met Met Leu Ala Glu 725 730 735 cag ttc aag cag aag gcc ctc ggc ctc
tcg cga ggg ggc aag ccg gca 2256 Gln Phe Lys Gln Lys Ala Leu Gly
Leu Ser Arg Gly Gly Lys Pro Ala 740 745 750 atc gtt cca gac aaa gag
gtg ttg tat caa caa tac gat gag atg gaa 2304 Ile Val Pro Asp Lys
Glu Val Leu Tyr Gln Gln Tyr Asp Glu Met Glu 755 760 765 gag tgc tca
caa gct gcc cca tat atc gaa caa gct cag gta ata gct 2352 Glu Cys
Ser Gln Ala Ala Pro Tyr Ile Glu Gln Ala Gln Val Ile Ala 770 775 780
cac cag ttc aag gaa aaa gtc ctt gga ttg atc gat aat gat caa gtg
2400 His Gln Phe Lys Glu Lys Val Leu Gly Leu Ile Asp Asn Asp Gln
Val 785 790 795 800 gtt gtg act cct gac aaa gaa atc tta tat gag gcc
ttt gat gag atg 2448 Val Val Thr Pro Asp Lys Glu Ile Leu Tyr Glu
Ala Phe Asp Glu Met 805 810 815 gaa gaa tgc gcc tcc aaa gcc gcc ctc
att gag gaa ggg cag cgg atg 2496 Glu Glu Cys Ala Ser Lys Ala Ala
Leu Ile Glu Glu Gly Gln Arg Met 820 825 830 gcg gag atg ctc aag tct
aag ata caa ggc ctc ctc ggg ata ctg cgc 2544 Ala Glu Met Leu Lys
Ser Lys Ile Gln Gly Leu Leu Gly Ile Leu Arg 835 840 845 cgg cac gtt
ggt cct ggc gag ggg gca gtg cag tgg atg aac cgg ctg 2592 Arg His
Val Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu 850 855 860
ata gcc ttc gcc tcc aga ggg aac cat gtt tcc ccc acg cac tac gtt
2640 Ile Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr
Val 865 870 875 880 ccg tct aga tcc cgg aga ttc gcc cag gcc ctg ccc
gtt tgg gcg cgg 2688 Pro Ser Arg Ser Arg Arg Phe Ala Gln Ala Leu
Pro Val Trp Ala Arg 885 890 895 ccg gac tat aac ccc ccg cta gtg gag
acg tgg aaa aag ccc gac tac 2736 Pro Asp Tyr Asn Pro Pro Leu Val
Glu Thr Trp Lys Lys Pro Asp Tyr 900 905 910 gaa cca cct gtg gtc cac
ggc aga tct tct cgg aga ttc gcc cag gcc 2784 Glu Pro Pro Val Val
His Gly Arg Ser Ser Arg Arg Phe Ala Gln Ala 915 920 925 ctg ccc gtt
tgg gcg cgg ccg gac tat aac ccc ccg cta gtg gag acg 2832 Leu Pro
Val Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu Thr 930 935 940
tgg aaa aag ccc gac tac gaa cca cct gtg gtc cat ggc aga aag acc
2880 Trp Lys Lys Pro Asp Tyr Glu Pro Pro Val Val His Gly Arg Lys
Thr 945 950 955 960 aaa cgt aac acc aac cgg cgg ccg cag gac gtc aag
ttc ccg ggt ggc 2928 Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val
Lys Phe Pro Gly Gly 965 970 975 ggt cag atc gtt ggt cgc agg ggc cct
cct atc ccc aag gct cgt cgg 2976 Gly Gln Ile Val Gly Arg Arg Gly
Pro Pro Ile Pro Lys Ala Arg Arg 980 985 990 ccc gag ggc agg acc tgg
gct cag ccc ggt tac cct tgg ccc ctc tat 3024 Pro Glu Gly Arg Thr
Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr 995 1000 1005 ggc aat
aag gac aga cgg tct aca ggt aag tcc tgg ggt aag cca ggg 3072 Gly
Asn Lys Asp Arg Arg Ser Thr Gly Lys Ser Trp Gly Lys Pro Gly 1010
1015 1020 tac cct tgg cca aga aag acc aaa cgt aac acc aac cga cgg
ccg cag 3120 Tyr Pro Trp Pro Arg Lys Thr Lys Arg Asn Thr Asn Arg
Arg Pro Gln 1025 1030 1035 1040 gac gtc aag ttc ccg ggt ggc ggt cag
atc gtt ggt cgc agg ggc cct 3168 Asp Val Lys Phe Pro Gly Gly Gly
Gln Ile Val Gly Arg Arg Gly Pro 1045 1050 1055 cct atc ccc aag gct
cgt cgg ccc gag ggc agg acc tgg gct cag ccc 3216 Pro Ile Pro Lys
Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro 1060 1065 1070 ggt
tac cct tgg ccc ctc tat ggc aat aag gac aga cgg tct acc ggt 3264
Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Lys Asp Arg Arg Ser Thr Gly
1075 1080 1085 aag tcc tgg ggt aag cca ggg tat cct tgg ccc 3297 Lys
Ser Trp Gly Lys Pro Gly Tyr Pro Trp Pro 1090 1095 4 1099 PRT
Artificial Sequence Description of Artificial Sequence MEFA 7.1 4
Met Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln 1 5
10 15 Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys
Val 20 25 30 Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly
Phe His Val 35 40 45 His Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr
Ser Ala Gly Pro His 50 55 60 Phe Asn Pro Leu Ser Arg Lys His Gly
Gly Pro Lys Asp Glu Glu Arg 65 70 75 80 His Val Gly Asp Leu Gly Asn
Val Thr Ala Asp Lys Asp Gly Val Ala 85 90 95 Asp Val Ser Ile Glu
Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys 100 105 110 Ile Ile Gly
Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly 115 120 125 Lys
Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg 130 135
140 Leu Ala Cys Gly Val Ile Gly Ile Ala Gln Asn Leu Asn Ser Gly Cys
145 150 155 160 Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg
Met Ala Trp 165 170 175 Lys Leu Gly Ser Ala Ala Arg Thr Thr Ser Gly
Phe Val Ser Leu Phe 180 185 190 Ala Pro Gly Ala Lys Gln Asn Glu Thr
His Val Thr Gly Gly Ala Ala 195 200 205 Ala Arg Thr Thr Ser Gly Leu
Thr Ser Leu Phe Ser Pro Gly Ala Ser 210 215 220 Gln Asn Ile Gln Leu
Ile Val Asp Phe Ile Pro Val Glu Asn Leu Glu 225 230 235 240 Thr Thr
Met Arg Ser Pro Val Phe Thr Asp Asn Ser Ser Pro Pro Val 245 250 255
Val Pro Gln Ser Phe Gln Val Ala His Leu His Ala Pro Thr Gly Ser 260
265 270 Gly Lys Ser Thr Lys Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr
Lys 275 280 285 Val Leu Val Leu Asn Pro Ser Val Ala Ala Thr Leu Gly
Phe Gly Ala 290 295 300 Tyr Met Ser Lys Ala His Gly Ile Asp Pro Asn
Ile Arg Thr Gly Val 305 310 315 320 Arg Thr Ile Thr Thr Gly Ser Pro
Ile Thr Tyr Ser Thr Tyr Gly Lys 325 330 335 Phe Leu Ala Asp Gly Gly
Cys Ser Gly Gly Ala Tyr Asp Ile Ile Ile 340 345 350 Cys Asp Glu Cys
His Ser Thr Asp Ala Thr Ser Ile Leu Gly Ile Gly 355 360 365 Thr Val
Leu Asp Gln Ala Glu Thr Ala Gly Ala Arg Leu Val Val Leu 370 375 380
Ala Thr Ala Thr Pro Pro Gly Ser Val Thr Val Pro His Pro Asn Ile 385
390 395 400 Glu Glu Val Ala Leu Ser Thr Thr Gly Glu Ile Pro Phe Tyr
Gly Lys 405 410 415 Ala Ile Pro Leu Glu Val Ile Lys Gly Gly Arg His
Leu Ile Phe Cys 420 425 430 His Ser Lys Lys Lys Cys Asp Glu Leu Ala
Ala Lys Leu Val Ala Leu 435 440 445 Gly Ile Asn Ala Val Ala Tyr Tyr
Arg Gly Leu Asp Val Ser Val Ile 450 455 460 Pro Thr Ser Gly Asp Val
Val Val Val Ala Thr Asp Ala Leu Met Thr 465 470 475 480 Gly Tyr Thr
Gly Asp Phe Asp Ser Val Ile Asp Cys Asn Thr Cys Val 485 490 495 Thr
Gln Thr Val Asp Phe Ser Leu Asp Pro Thr Phe Thr Ile Glu Thr 500 505
510 Ile Thr Leu Pro Gln Asp Ala Val Ser Arg Thr Gln Arg Arg Gly Arg
515 520 525 Thr Gly Arg Gly Lys Pro Gly Ile Tyr Arg Phe Val Ala Pro
Gly Glu 530 535 540 Arg Pro Ser Gly Met Phe Asp Ser Ser Val Leu Cys
Glu Cys Tyr Asp 545 550 555 560 Ala Gly Cys Ala Trp Tyr Glu Leu Thr
Pro Ala Glu Thr Thr Val Arg 565 570 575 Leu Arg Ala Tyr Met Asn Thr
Pro Gly Leu Pro Val Cys Gln Asp His 580 585 590 Leu Glu Phe Trp Glu
Gly Val Phe Thr Gly Leu Thr His Ile Asp Ala 595 600 605 His Phe Leu
Ser Gln Thr Lys Gln Ser Gly Glu Asn Leu Pro Tyr Leu 610 615 620 Val
Ala Tyr Gln Ala Thr Val Cys Ala Arg Ala Gln Ala Pro Pro Pro 625 630
635 640 Ser Trp Asp Gln Met Trp Lys Cys Leu Ile Arg Leu Lys Pro Thr
Leu 645 650 655 His Gly Pro Thr Pro Leu Leu Tyr Arg Leu Gly Ala Val
Gln Asn Glu 660 665 670 Ile Thr Leu Thr His Pro Val Thr Lys Tyr Ile
Met Thr Cys Met Ser 675 680 685 Ala Asp Leu Glu Val Val Thr Ser Ala
Cys Ser Gly Lys Pro Ala Ile 690 695 700 Ile Pro Asp Arg Glu Val Leu
Tyr Arg Glu Phe Asp Glu Met Glu Glu 705 710 715 720 Cys Ser Gln His
Leu Pro Tyr Ile Glu Gln Gly Met Met Leu Ala Glu 725 730 735 Gln Phe
Lys Gln Lys Ala Leu Gly Leu Ser Arg Gly Gly Lys Pro Ala 740 745 750
Ile Val Pro Asp Lys Glu Val Leu Tyr Gln Gln Tyr Asp Glu Met Glu 755
760 765 Glu Cys Ser Gln Ala Ala Pro Tyr Ile Glu Gln Ala Gln Val Ile
Ala 770 775 780 His Gln Phe Lys Glu Lys Val Leu Gly Leu Ile Asp Asn
Asp Gln Val 785 790 795 800 Val Val Thr Pro Asp Lys Glu Ile Leu Tyr
Glu Ala Phe Asp Glu Met 805 810 815 Glu Glu Cys Ala Ser Lys Ala Ala
Leu Ile Glu Glu Gly Gln Arg Met 820 825 830 Ala Glu Met Leu Lys Ser
Lys Ile Gln Gly Leu Leu Gly Ile Leu Arg 835 840 845 Arg His Val Gly
Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu 850 855 860 Ile Ala
Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val 865 870 875
880 Pro Ser Arg Ser Arg Arg Phe Ala Gln Ala Leu Pro Val Trp Ala Arg
885 890 895 Pro Asp Tyr Asn Pro Pro Leu Val Glu Thr Trp Lys Lys Pro
Asp Tyr 900 905 910 Glu Pro Pro Val Val His Gly Arg Ser Ser Arg Arg
Phe Ala Gln Ala 915 920 925 Leu Pro Val Trp Ala Arg Pro Asp Tyr Asn
Pro Pro Leu Val Glu Thr 930 935 940 Trp Lys Lys Pro Asp Tyr Glu Pro
Pro Val Val His Gly Arg Lys Thr 945 950 955 960 Lys Arg Asn Thr Asn
Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly 965 970 975 Gly Gln Ile
Val Gly Arg Arg Gly Pro Pro Ile Pro Lys Ala Arg Arg 980 985 990 Pro
Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr 995
1000 1005 Gly Asn Lys Asp Arg Arg Ser Thr Gly Lys Ser Trp Gly Lys
Pro Gly 1010 1015 1020 Tyr Pro Trp Pro Arg Lys Thr Lys Arg Asn Thr
Asn Arg Arg Pro Gln 1025 1030 1035 1040 Asp Val Lys Phe Pro Gly Gly
Gly Gln Ile Val Gly Arg Arg Gly Pro 1045 1050 1055 Pro Ile Pro Lys
Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro 1060 1065 1070 Gly
Tyr Pro Trp Pro Leu Tyr Gly Asn Lys Asp Arg Arg Ser Thr Gly 1075
1080 1085 Lys Ser Trp Gly Lys Pro Gly Tyr Pro Trp Pro 1090 1095 5
21 PRT Artificial Sequence Description of Artificial Sequence
consensus sequence 5 Gly Ser Ala Ala Arg Thr Thr Ser Gly Phe Val
Ser Leu Phe Ala Pro 1 5 10 15 Gly Ala Lys Gln Asn 20 6 10 DNA
Artificial Sequence Description of Artificial Sequence ligated DNA
piece 6 acaaaacaaa 10 7 23 PRT Artificial Sequence Description of
Artificial Sequence NS4A peptide 7 Lys Lys Gly Ser Val Val Ile Val
Gly Arg Ile Val Leu Ser Gly Lys 1 5 10 15 Pro Ala Ile Ile Pro Lys
Lys 20
* * * * *